Michael V. Sofroniew - US grants

DESCRIPTION (provided by applicant): Astrocytes respond to spinal cord injury (SCI) by altered gene expression, hypertrophy and proliferation, a process known as reactive astrocytosis. Considerable descriptive information is available about molecules produced by reactive astrocytes, but the functions of these cells are not well understood. Astrocyte scars are thought to prevent axon regeneration after SCL but evidence also points towards important potential roles for reactive astrocytes in the process of acute local tissue repair. We have developed an experimental model to demonstrate specific functions of reactive astrocytes by targeting the specific ablation of this cell type after CNS injury in transgenic mice. Using these mice we have shown that after injury in the forebrain, reactive astrocytes are essential for protection of local neurons, repair of the blood brain barrier and restricting the infiltration of inflammatory white blood cells. In addition, we found increased local sprouting of axons in areas where reactive astrocytes were ablated. In this proposal we will apply this transgenic model to study the roles of reactive astrocytes in SCI. First, we will ablate reactive scar-forming astrocytes at the site of a small, acute spinal cord lesion to characterize the roles played by these cells in (i) the acute injury response and repair process after SCI, and (ii) long term effects on nerve fiber tract integrity and capacity for regrowth. These findings will tell us whether reactive, scar-forming astrocytes are, as we predict, essential for acute tissue repair after SCI, such that their absence or dysfunction will markedly exacerbate the tissue degeneration and detrimental effects associated with small spinal cord lesions. Next, we will determine the extent of axon survival and capacity for local and long distance axon regeneration in the transected dorsal columns of the spinal cord, after transgenically-targeted ablation of reactive scar-forming astrocytes, alone or in combination with grafts of immature astrocytes, neural stem cells or olfactory ensheathing cells. These findings will tell us whether ablation and appropriate replacement of reactive, scar-forming astrocytes can, as we predict, lead to substantially improved regrowth of transected long tract axons after acute SCI. Together, information from these studies will provide essential ground work for future dissection of the underlying molecular signaling mechanisms that mediate specifically identified roles (both beneficial and detrimental) of reactive astrocytes after SCI.

After a wide variety of spinal cord injuries (SCI), humans and animals can recover a greater level of functional stepping if they are given appropriate use-dependent step training. Following complete SCI, substantial reorganization of sensorimotor pathways occurs caudal to the lesion. Step training significantly influences this reorganization. Behavioral and physiological effects of SCI and step training are reflected in adaptations in most, if not all, intrinsic spinal neurotransmitter systems. Previous studies in this program have characterized extensive neuromuscular plasticity following complete SCI. Little is known about the potential for interactions of intrinsic spinal systems involved in neuromuscular plasticity with descending axons that are spared after partial SCI or are regenerating after experimental interventions. The present study will examine the contribution of different descending spinal pathways to the control of stepping and determine how lesions of different pathways influence neuromuscular plasticity after SCI and step training. Understanding which descending spinal pathways are important for control of specific aspects of hindlimb movement, and which pathways should be particularly targeted for regeneration, would represent an important advance. In addition, we will study the effects of partial axon regeneration achieved after transgenically targeted ablation of scar forming, reactive astrocytes. We hypothesize that partial regeneration of descending pathways will interact synergistically with the spinal neuromuscular plasticity that is induced by step training and will augment control of stepping after SCI. These studies will take advantage of recently developed, robot-assisted evaluation of stepping, and combine this with video analysis and electromyographic recordings in adult transgenic mice. Mice will be used as experimental animals because transgenic technology in mice provides a powerful means for precise cellular and molecular manipulations whose effects can be evaluated at the systems level in vivo. This technology holds considerable promise for dissecting out specific molecular and cellular mechanisms after SCI. Results from the present study will establish a framework for quantitative evaluation of the neuromuscular control of stepping in mice, and will provide important information about (i) how lesions of different spinal pathways influence neuromuscular plasticity and the control of stepping, (ii) how partial axon regeneration may improve this control, and (iii) how step training augments these processes.

DESCRIPTION (provided by applicant): Substantial evidence indicates that the birth of new neurons (neurogenesis) continues throughout life in specific regions of the adult brain, including humans. The biology of the adult neural progenitors that give rise to new neurons is of considerable interest reflecting not only their potential functional roles, but also their potential for treatment or repair of neurological illness or injury. The identity and origin of adult neural progenitors are controversial. Experimental evidence suggests that some adult neural progenitors express glial fibrillary acidic protein (GFAP) and exhibit certain characteristics of astroglia. These observations raise questions and challenges. Given the fundamental roles of GFAP expressing astroglia in neural injury, disease and repair, it is important to understand the relationships, if any, between astroglia and adult neural progenitors. We have a longstanding interest in GFAP-expressing astroglia, and have developed transgenic mouse models to study these cells in neural injury and repair. Here, we apply these models to determine (i) the relative contribution, if any, of GFAP-expressing cells to adult neurogenesis in vivo and in vitro, (ii) whether all GFAP-expressing glia have neurogenic potential or whether this potential is associated with a subpopulation of cells that exhibits distinct phenotypic characteristics, and (iii) factors that regulate the neurogenic potential of GFAP-expressing neural progenitors, in particular after brain injury. To do so we use in vitro and in vivo techniques and several transgenic mouse models that allow (a) ablation of GFAP-expressing cells, (b) lineage analysis and fate mapping of progeny of GFAP-expressing cells, and (c) deletion of genes specifically from GFAP-expressing cells. Our preparatory work and preliminary data are consistent with several hypotheses including: (1) the predominant neural progenitors in adult forebrain express GFAP; (2) not all GFAP-expressing glia have neurogenic potential, neurogenic potential correlates with the presence of GFAP-expressing cells that exhibit certain phenotypic characteristics similar to radial glia; (3) the multipotent potential of GFAP-expressing progenitors can be manipulated by environmental conditions. Findings from studies proposed here will contribute fundamental information towards establishing the identity and regulation of adult neural progenitor cells, and towards defining the relationships between neural progenitors and the astroglia that respond to injury and disease. Understanding the biology of GFAP-expressing neural progenitors and their relationship to astroglia, which are widespread throughout the central nervous system, may reveal novel research avenues towards improving neural repair after injury or disease.

DESCRIPTION (provided by applicant): All central nervous system (CNS) insults including trauma, infection, ischemia and degenerative disease trigger changes in astroglia known as reactive astrogliosis. The roles of reactive astroglia are not well established. Astroglia stimulated in vitro can produce a wide variety of molecules including both pro- and anti-inflammatory regulators, as well as cytotoxic and neuroprotective molecules. Accordingly, both harmful and beneficial effects have been attributed to reactive astrocytes. Our central hypothesis is that during the response to CNS insults, reactive astrocytes can exert effects that may be either beneficial or detrimental to clinical outcome in a manner that is context dependent and is regulated by specific inter- and intra-cellular signaling mechanisms. The signaling mechanisms that regulate activities implemented by reactive astrocytes in response to specific situations in vivo are not well understood. Our previous work used a transgenic mouse model to ablate reactive astrocytes and showed that these cells play pivotal roles in restricting inflammation and protecting tissue after brain or spinal cord injury in vivo. Our next goal is to identify molecular mechanisms that regulate specific activities of reactive astrocytes. To do so we have developed conditional gene deletion or knockout technology (CKO) for astrocytes using the Cre/loxP system under regulation of the mouse GFAP promoter in transgenic mice. Here we propose to determine the effects of selectively deleting STAT3, an intracellular signal transducer that has been implicated as a regulator of reactive astrogliosis. We will study spinal cord injury (SCI) and in vitro preparations using a combination of quantitative morphological and biochemical analyses. Our preliminary data show that mice with astroglial STAT3-CKO have CNS of normal size and cytology, and that astrocytes are generated in normal numbers. After SCI, reactive astrogliosis is attenuated and scar formation is disrupted in mice with astroglial STAT3- CKO. This proposal builds on our preliminary findings by investigating three specific aims that will determine the effects of astroglial STAT3-CKO: (1) on quantitative measures of astrocyte reactivity and scar formation after SCI in vivo and on various regulatory signaling pathways in vitro;(2) on inflammation, lesion size and short-term motor behavior after SCI in vivo, and on astrocyte expression in vitro of molecules that influence inflammation and cytotoxicity;and (3) on axon regeneration, inflammation and long-term motor behavior after SCI in vivo, and on the production in vivo and in vitro of molecules that inhibit both axon regeneration and inflammatory cell migration. The findings will provide fundamental information about signaling mechanisms that regulate astrogliosis after SCI. Such mechanistic information is essential for understanding the cellular and molecular interactions that determine functional outcome after SCI, and will help to identify key pathways and molecules that warrant targeting for potential therapeutic manipulation. PUBLIC HEALTH RELEVANCE: Spinal cord injury has devastating consequences and little or no treatment options. Scar formation by reactive astrocytes is a prominent feature of spinal cord injury, and both harmful and beneficial effects have been attributed to reactive astrocytes. The work proposed here will benefit public health by identifying molecular signaling mechanisms that regulate specific functions of reactive astrocytes after spinal cord injury and can be targeted for therapeutic manipulation to improve outcome.

DESCRIPTION (provided by applicant): Astrocytes play well documented supportive roles in the brain. In addition, emerging roles for astrocytes include signaling to and from neurons, and regulation of local blood flow. Certain astrocyte functions are correlated with, or regulated by, cytosolic calcium transients, which are considered a physiological signal reflecting astrocyte excitability. Astrocytes interact with neurons and blood vessels primarily with their distal processes, but it is currently not possible to measure calcium signals non-invasively in astrocyte processes either in vitro in tissue slice preparations or in vivo. A method for measuring calcium in astrocyte processes in slices and in vivo would enable the rigorous testing of mechanistic hypotheses regarding astrocyte roles in brain function in the healthy CNS, and would open up new ways to study the impact of reactive astrogliosis, which occurs in response to all forms of injury and disease, on these same CNS functions. To develop such a method, we have modified a genetically encoded calcium sensor called GCaMP2 to carry a membrane tethering domain (called Lck) on its N terminus, thus generating Lck-GCaMP2. Our findings thus far show that Lck-GCaMP2 allows the non-invasive imaging of calcium levels in astrocytes near the membrane and in processes in cell cultures. This level of resolution is possible because the Lck-GCaMP2 is selectively and highly expressed in the plasma membrane, providing micrometer scale spatial information. This approach will help reveal when, where and how astrocytes are activated during physiological and pathophysiological processes. In this proposal we seek to exploit our new approach and provide important new resources for the astrocyte signaling community by generating and characterizing novel transgenic mice that will allow calcium imaging in astrocyte processes in tissue slices and in vivo. We have two specific aims. In Aim 1 we will manufacture gene constructs that target Lck-GCaMP2 to astrocytes and use these constructs to establish founder lines of transgenic mice. In Aim 2 we will characterize and use these Lck- GCaMP2 transgenic mice to study mechanisms and functions of calcium signaling in astrocyte processes in hippocampal slices. We will test two hypotheses: (i) that astrocytes display spatially compartmentalized calcium signaling and (ii) that TGF2 has direct, receptor mediated effects on calcium signaling in the processes of reactive astrocytes, thereby providing a mechanism through which reactive astrogliosis could influence neurons. The work proposed here will provide novel, well characterized optical reporter mice that will allow us and others to measure precisely localized calcium signals in astrocyte somata and processes within intact tissue structures such as brain slices and in vivo. These new reporter mice will be valuable general tools for the astrocyte community by allowing researchers to measure local astrocyte calcium signals in processes that are currently inaccessible to conventional imaging methods. PUBLIC HEALTH RELEVANCE: We will develop mouse models that will allow us and other researchers to monitor and track calcium signaling in astrocytes in vitro and in vivo. The availability of these mice would constitute exceptional tools with which to study the role of astrocytes in the normal healthy brain and in diseases of the nervous system, including epilepsy and neurodegeneration as well as during brain injury and repair.

Technical: There is a need for polymeric drug carriers that can be prepared using a versatile method that allows fine tuning of chemical composition and structure, and use building blocks that are biocompatible and easily functionalized. The goal of this project is to develop and study multifunctional amphiphilic block copolypeptides containing modified poly(L-methionine) segments, MMOD, that can be assembled into vehicles for intracellular drug delivery. Recent synthetic advances in this lab now allow the development of entirely new polypeptide amphiphiles utilizing MMOD domains that are designed so that individual segments can play unprecedented multiple functional roles in the resulting nanocarrier assemblies. This innovative approach provides a new method for introducing functionality into polymeric nanocarriers and will develop and test a new class of methionine based biomaterials. The incorporation of methionine segments and their subsequent modification is a straightforward, scalable process, and allows unprecedented control in the ability to add complex functionality and biological activity to polypeptides. Some MMOD residues also occur naturally in biological systems and these will be used strategically to promote release of therapeutics, and may also provide other therapeutic benefits. The MMOD segments will be utilized as new, functional hydrophilic domains capable of providing multiple combinations of solubility, biocompatibility, therapeutic binding, cell uptake, enzyme-response, pH response, and chemoselective bioconjugation. Specifically, the project will design, prepare, and characterize vesicle forming block copolypeptides containing MMOD segments as carriers for therapeutics with low cytotoxicity and capability for cell uptake, endosomal release and intracellular carrier disruption. In addition, it will test the capabilities of these carriers using in vitro cell culture and trafficking studies. The knowledge gained from these studies will allow fine tuning of carrier properties for downstream specific uses in encapsulation and delivery of drugs, and will lay groundwork for development of a new class of functional biomaterials for medical applications.

Non-Technical: In this project, the PIs will continue their successful inclusion of underrepresented groups, teaching and training of graduate and undergraduate students, and dissemination of their research findings in publications and presentations. Some examples of these efforts from the previous grant period are: development and improvement of bioengineering courses incorporating concepts from the project such as intracellular trafficking and bioconjugation methods; recruitment of a Hispanic female student (Ph.D. granted in March 2013) and an African American female student for this project (1st year); PI and student presentations of research results at national and local meetings (ACS, BMES, MRS, Society for Advancement of Hispanics, Chicanos, and Native Americans in Science (SACNAS) national meeting); and presentations incorporating this research by the PI to encourage students to pursue careers in science (2010 UCSB Summer School on materials synthesis; 2011 NAE Grand Challenges Summit for graduate students; 2012 International Young Scientist Symposium, Bordeaux, France). Professor Kamei has also made annual visits to elementary and high schools in East Los Angeles (one is 90% Hispanic) to inspire youth in this system to become scientists and engineers. Ph.D. students trained under this program are valuable in the industrial job force (both pharmaceutical and materials science areas) since they will learn fundamentals of polymer synthesis using catalysis and self-assembly, cell culture and drug trafficking, as well as more applied areas of materials characterization and property evaluation. These undergraduate students have also done well by being admitted into prestigious Ph.D. (Washington, MIT, UCSB) and MD (Cornell, Texas A&M) programs, and obtaining NSF graduate fellowships.

DESCRIPTION (provided by applicant): An important strategy for improving outcome after spinal cord injury (SCI) is to achieve axon regrowth across lesions to reach functional neural targets. Various molecules have the potential to foster axon regrowth but cannot pass the blood brain barrier and exhibit activity in many central nervous system (CNS) regions, necessitating local delivery to achieve efficacy while avoiding side effects. Prolonged but temporary delivery is needed. Clinically translatable methods for such delivery are lacking. Our goal is to develop functionalized diblock copolypeptide hydrogels (DCH) as fully synthetic biomaterials that that can easily and safely be injected into, and near, SCI lesions to provide depots for sustained local release of multiple molecules that manipulate local cells and stimulate axons to regrow into healthy tissue. Our previous works demonstrates DCH safety and efficacy to deliver growth factors that exert predictable effects over distances of several mm in CNS. New preliminary data show that: (1) DCH depots injected 2 days after SCI are able to simultaneously deliver multiple growth factors that stimulate substantive regrowth of both sensory and propriospinal fibers throughout the SCI lesion core. We find that these regrowing axons track along cells with newly upregulated laminin expression, and that regrowth can be blocked by simultaneous delivery of function-blocking antibodies that disrupt laminin-integrin binding. (2) When DCH delivery of multiple growth factors is combined with attenuation of glial scar by deletion of STAT3 in transgenic mice, axons regrow beyond the lesion core into the distal glial scar. New data also show DCH can deliver hydrophobic small molecules like JSI, which inhibits STAT3 and attenuates scar formation in a manner comparable to our transgenic mice. (3) When multiple DCH depots are placed into both the lesion core and distal healthy tissue, we find considerable axon regrowth into healthy tissue areas that contains viable NeuN-positive neurons. The work proposed will build on these preliminary findings and use DCH depots injected after SCI to simultaneously deliver different types of molecules (including multiple protein growth factors, antibodies and small hydrophobic molecules that manipulate gene expression) in order to: (i) manipulate cells in scar and lesion core to enable and support axon regrowth, (ii) directly stimulate and guide axon regrowth into, through and beyond lesions into healthy tissue, (iii) dissect cellular and molecular mechanisms that underlie the axon regrowth stimulated by different molecules or combinations of molecules, and (iv) test whether regrowing propriospinal neurons that reach healthy tissue are able to contact neurons there and are able to form relay connections that improve locomotor function. This DCH depot approach will provide a powerful tool for the experimental investigation of cellular and molecular mechanisms after SCI, and will facilitate the extensive trial and error testing needed to identify appropriate molecules for potential clinical translation. In addition, because DCH are fully synthetic biomaterials, there is also a realistic potential for clinical translation of DCH for use in SCI.

Anatomically complete spinal cord injury (SCI) transects and eliminates all functional connections across the level of the lesion, and in adults, axons fail to regrow spontaneously across such lesions. Restoring voluntary control of function will require interventions to establish new neural connections across the lesion. During the previous funding cycle of this grant, we identified a mechanism-based biological repair strategy for achieving robust regrowth of propriospinal axons across complete SCI lesions in rodents. We showed that providing three mechanisms essential for axon growth during development, (i) neuron intrinsic growth capacity, (ii) growth-supportive substrate and (iii) chemoattraction, can achieve robust regrowth of axons through and beyond anatomically complete SCI. This axon regrowth was 100-fold greater than controls, passed a full spinal segment beyond the injuries, and was able to restore significant electrophysiological conduction capacity across injuries. To achieve the spatially and temporally controlled in vivo molecular delivery required to realize this axon regrowth, we engineered biomaterial depots that enabled us to mimic certain spatiotemporal events regulating axon growth during development. In the project proposed here, we will build on this work and use our newly developed synthetic hydrogel vehicle to deliver molecules that direct the differentiation in vivo of grafted neural progenitor cells (NPC) into axon-supportive immature astroglia that repopulate non-neural lesion cores and reestablish a multicellular neural environment favorable for long term support of host propriospinal axons chemoattracted to regrow through lesions into spared neural tissue. Our hypothesis is that repopulating (and ?reneuralizing?) such non-neural lesion cores, or their cysts, with immature astroglia will promote long-term axonal maintenance and provide a favorable niche for remyelinating cells. Our objective is to develop engineering approaches that facilitate doing so. Our premise is that our hydrogel vehicles can deliver both: (i) molecules that direct the differentiation of NPC in vivo, and (ii) molecules that chemoattract host axons. Our past work and preliminary data show that NPC grafted in our hydrogel vehicles are good candidates to generate support cells for host axons as well as for host-derived oligodendrocyte progenitor cells that migrate into areas of grafted cells. We have also shown that propriospinal neurons are good targets for bridging host axons across complete SCI lesions into spared neural tissue below injuries. The work for this proposal will advance the development of mechanism- based engineering approaches to repair neural tissue after severe SCI, stroke and other CNS disorders with large focal lesions.