Binhai Zheng - US grants

DESCRIPTION (provided by applicant): Axon regeneration failure in the adult mammalian central nervous system (CMS) has been attributed at least in part to the inhibitory nature of the CNS myelin. Three "classical" myelin-derived neurite outgrowth inhibitors, Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp), have been identified that exhibit potent inhibitory activity on neurite outgrowth in vitro. However, the central question remains as to the contribution of these inhibitors to CNS axon regeneration failure in vivo. Our central hypothesis is that myelin-derived neurite growth inhibitors Nogo, MAG and OMgp play a significant and potentially redundant role in blocking spinal axon regeneration. The overall approach is to examine axon regeneration in the spinal cord of mice with gene deletion in one or more myelin inhibitors chronically or acutely, specifically addressing the issues of developmental compensation and functional redundancy. By acute gene deletion, we address the issue of developmental compensation in germline mutants. By deleting the three inhibitors simultaneously, we address the issue of functional redundancy. Aim 1. To assess the role of Nogo and OMgp in regeneration failure by examining the effect of acutely deleting Nogo or OMgp on corticospinal and raphespinal serotonergic fiber regeneration. We will employ an inducible knockout system to acutely delete Nogo or OMgp in oligodendrocytes and then examine the regenerative response of the corticospinal tract (CST) and the raphespinal serotonergic fiber tract. Aim 2. To assess the combined contribution of Nogo, MAG and OMgp in CNS axon regeneration failure by ascertaining the regeneration potential of the corticospinal and raphespinal serotonergic fiber tracts in mice deficient in all three inhibitors. Aim 3. To test whether increasing the intrinsic growth potential has a synergistic effect with removing myelin inhibitors in promoting spinal axon regeneration. Previous studies indicate that a conditioning lesion to the peripheral branch of the dorsal root ganglion (DRG) neurons augments the intrinsic growth potential of the neurons and promotes the regeneration of the central branch in vivo. We will test whether this enhanced regeneration is further enhanced in Nogo/MAG/OMgp triple mutant, and whether there is a synergistic effect between deleting the three myelin inhibitors and a conditioning lesion. Together, these studies will provide important insight into the role of myelin inhibitors in spinal axon regeneration failure. Understanding the role of these myelin inhibitors in CNS axon regeneration is crucial to the design of any therapeutic intervention to promote axon regeneration and spinal cord repair targeting this group of molecules.

DESCRIPTION (provided by applicant): The poor regenerative ability of axons in the adult mammalian central nervous system (CNS) underlies the limited functional recovery following spinal cord injury, traumatic brain injury, white matter stroke and certain neurodegenerative disorders. There are two forms of injury-induced axonal growth that may contribute to functional repair: regeneration of injured axons and compensatory growth (or sprouting) of uninjured axons. The goal of this application is to gain a better understanding of the role of the molecular players - both extrinsic factors in the CNS environment and intrinsic factors in the neurons - in axon sprouting and regeneration. The study will focus on two inhibitors of axon growth made by the CNS myelin (Nogo, OMgp) and PTEN, a negative regulator of neuron-intrinsic growth potential. The overall approach is to study the responses of axons to injury in mice lacking one or more growth regulators so the normal function of these proteins can be assessed. Aim 1 will determine the cell type that is important for the role of Nogo in axon sprouting of the corticospinal tract (CST), whether Nogo and OMgp synergize to prevent CST axon sprouting, and will further explore the functional consequences of such enhanced sprouting in conjunction with rehabilitation. Aim 2 will determine the combined effect of targeting the myelin inhibitor(s) and PTEN on spinal axon sprouting and regeneration, and whether enhanced axonal growth leads to synapse formation and functional recovery. The proposal takes advantage of the power of mouse genetics to pinpoint the role of specific intrinsic and extrinsic regulators and will likely yield important insights on the functions of these molecules in injury-induced axonal growth. A better understanding of the molecular determinants of injury-induced axonal growth in the adult CNS is crucial to the design of effective therapeutic strategies for various neurological conditions including spinal cord injury.

DESCRIPTION (provided by applicant): In spinal cord injuries, axons are cut from the neuronal cell bodies, and lack of axon regeneration is the principal cause of no or limited functional recovery in the central nervous system (CNS, including the brain and the spinal cord). While earlier studies emphasized the importance of neuron-extrinsic mechanisms, recent development in the field highlighted the central role of neuron-intrinsic control of axon growth and regeneration after CNS injury. Meanwhile, it is increasingly clear that targeting one molecule or pathway at a time is unlikely to bring about functionally meaningful axon growth and regeneration. miRNAs are small non-coding RNAs that post-transcriptionally regulate protein synthesis. One miRNA can regulate the expression of multiple proteins in multiple pathways, sometimes related in function. Here we propose to explore the role of miRNAs in spinal axon sprouting and regeneration after CNS injury. We hypothesize that in neurons some miRNAs are growth inhibitory while others are growth promoting. Reversal of the expression of growth inhibitory and promoting miRNAs after CNS injury may allow neurons to enter a more regenerative state, thus promoting axon growth and regeneration. In Aim 1, we will systematically profile miRNA expression in corticospinal neurons and axons before and after spinal cord injury and compare these data to miRNA expression in postnatal neurons that still possess significant axon growth ability. A comparison of miRNA expression profiles in neurons of different levels of regenerative abilities may provide important clues to the pattern and identiy of the miRNAs that may positively or negative regulate axon growth. The expression profiling will be considered together with target predictions to narrow down the candidate miRNAs to be functionally tested. In Aim 2, we will assess the effect of manipulating candidate miRNAs by overexpression or inhibition on axon growth in vitro using microfluidic chambers. We will use adeno-associated virus (AAV) to deliver miRNAs or inhibitory sponge constructs. These experiments will help us to further narrow down the number of candidates for in vivo studies. In Aim 3, we will assess the effect of manipulating candidate miRNAs on corticospinal axon sprouting and regeneration using pyramidotomy and dorsal hemisection spinal cord injury respectively. Together, these studies will start to assess the role and therapeutic potential of miRNAs for promoting axonal repair after CNS injury. The unique feature of miRNAs regulating multiple molecular targets and pathways simultaneously renders them attractive therapeutic targets and tools for diseases and injuries. This proposal represents an early step in the application of miRNA biology to axonal repair after CNS injury.

? DESCRIPTION (provided by applicant): The limited axonal growth after central nervous system (CNS) injury is the principal cause of no or limited functional recovery after spinal cord injury in humans. While earlier studies emphasized the importance of neuron-extrinsic mechanisms, recent development in the field highlighted the central role of neuron-intrinsic control of axon growth and regeneration after CNS injury. Among neuron-intrinsic regulators, there are factors that sense and mediate the injury signals to elicit diverse responses in differen neuronal populations. Recent discoveries in model organisms such as the nematode and the fruit fly indicate a critical role for a MAP kinase kinase kinase (MAPKKK, or MAP3K), DLK, in axon degeneration and regeneration. There are two sequence homologues in mammals: DLK (also known as MAP3K12) and LZK (also known as MAP3K13). Several studies indicate that mammalian DLK/MAP3K12 is important in axonal responses to injury. Little is known about the other homologue LZK/MAP3K13. Our preliminary studies strongly indicate that LZK regulates axon growth. Here we test the hypothesis that LZK is an important regulator of axonal responses to injury in the mammalian nervous system. We will test the effect of loss of function and gain of function genetic manipulations of LZK in different injury models. In this context, we will also examine any functional redundancy or synergy between DLK and LZK by combined genetic manipulations. To this end, we have generated both loss and gain of function alleles for DLK and LZK in mice. We will examine injury-induced axon degeneration, regeneration (axonal growth from injured neurons) and sprouting (axonal growth from uninjured neurons) with a number of injury models in the CNS, supplemented by injury models in the PNS (peripheral nervous system). Finally, we will examine potential interaction between the LZK and DLK signaling pathways with other neuron- intrinsic (e.g. PTEN) regulators of axon growth. Together, these studies will reveal the endogenous role of the LZK and DLK signaling, and assess the therapeutic potential of activating LZK and DLK signaling in promoting axonal repair after CNS injury.

DESCRIPTION (provided by applicant): The ability to test hypotheses in a variety of neuroscience fields has exploded due to the new ability to monitor and manipulate key cellular events in living animals and other models of disease and neuronal function in a high throughput fashion using modern cell biological approaches. Our previous award provided the foundation for a world-class Neuroscience Core, in support of NINDS-funded aims of UCSD Neuroscientists. With this application, we seek to incorporate high-throughput and high-sensitivity approaches in our established Neuroscience Imaging Core through purchase and active management of new equipment for our Major Users. Furthermore, we aim to increase the number NINDS-funded investigators participating in this initiative from 8 to 28 labs, to reflect ou outstanding cellular neuroscience community and number of qualifying projects. The University of California, San Diego Neurosciences Core has grown to be a centerpiece for research within the neuroscience community. Its existence has resulted in remarkable yields in productivity, expanded scientific scope and ability to test hypotheses using cutting edge technology and in vivo approaches. In this application, we propose move into exciting new areas through: 1. In vivo brain imaging of live neural tissue with high-sensitivity multiphoton microscopy. 2. High throughput electron microscopic ultrastructural imaging taking advantage of the latest breakthroughs in serial block face scanning electron microscopy (SBFSEM) and emergent probe technologies developed at UCSD. 3. More than tripling the number of Major User NINDS-funded investigators at UCSD. The research that this equipment will support involves neuronal stem cell differentiation, migration, axonal guidance, activity-dependent plasticity, connectivity, injury repair, degenerative disease and developmental biology. The requested systems were chosen because of their high data quality, unsurpassed abilities to document events in the nervous system not previously possible, and their ease of use, which is critical for a multi-user Core. The Neuroscience Core offers on-hand technicians to provide training for each of the services provided. The Core leverages the expertise of the other UCSD efforts including the National Center for Microscopy and Imaging Research (NCMIR), which will provide expertise and staff to meet the proposed goals to support training and research. The flexibility, dynamic range, sensitivity, and processing capabilities that will be provided by these tools is essential fr the next phase of the NINDS-funded work that has important implications for multiple nervous system diseases.

PROJECT SUMMARY / ABSTRACT ? ADMINISTRATIVE CORE The University of California San Diego Neuroscience Microscopy Imaging Core is a microscopy imaging center operating as one core. As such, there is only one combined Project Summary / Abstract in this application. Please refer to ?Project Summary / Abstract ? Overall?.

PROJECT SUMMARY / ABSTRACT ? SCIENTIFIC CORE The University of California San Diego Neuroscience Microscopy Imaging Core is a microscopy imaging center operating as one core. As such, there is only one combined Project Summary / Abstract in this application. Please refer to ?Project Summary / Abstract ? Overall?.

The ability to test hypotheses in a variety of neuroscience fields has expanded exponentially due to new and emerging technologies that detect and probe molecular, cellular and physiological events in terminally collected samples or living model organisms with ever increasing power and capabilities. Supported by an NINDS P30 grant since 2003, the University of California San Diego Neuroscience Microscopy Imaging Core has grown into a world-class core and a centerpiece for local neuroscience research. Importantly, the Core serves an otherwise unmet neuroscience research need in microscopy imaging of many laboratories. Its existence has resulted in remarkable yields in productivity, expanded scientific scope and ability to test hypotheses using cutting edge technologies. In this application, we seek to acquire a Zeiss Lightsheet Z.1 microscope for fast, gentle, multiview imaging deep into thick samples such as cleared brain and spinal cord tissue blocks or living organisms. The requested system was chosen because of the high quality of data that it generates, unsurpassed abilities to document events in the nervous system not previously feasible and its ease of use, which is critical for a multi-user core. In addition, we ask for funds to help maintain, operate and support a number of existing cutting edge imaging tools at the Core. On top of 35 NINDS-funded Major User labs with 44 qualifying projects, a wider base of neuroscience investigators benefit from access to our Core. UCSD is a leading institution in neuroscience research, with our neuroscience graduate program consistently ranked among the top in the nation. The strong support of local neuroscience researchers along with that of institutional leadership, the synergies and the economies of scale that the Core has created and the continued P30 grant support will ensure the future success of our Core. In return, our Core supports the mission of the NINDS by serving a wide range of outstanding research programs that aim to gain fundamental knowledge of the nervous system and to reduce the burden of neurological diseases.

PROJECT SUMMARY / ABSTRACT After spinal cord injury, a plethora of cellular responses impact functional recovery. Neurons may be preserved or undergo cell death, axon degeneration and/or regenerative attempt. Astrocytes may become hypertrophic, seal off the injury epicenter and influence axonal response in complex ways. Other cell types such as fibroblasts/pericytes, microglia, macrophages also play important roles. Understanding how different cell types respond to injury, how their responses are regulated and how they contribute to functional recovery is critical for developing therapeutic intervention to promote functional repair after spinal cord injury. In the past, our lab has mostly focused on the molecular control of neuronal responses to injury, and in particular axon regeneration and sprouting following CNS injury. Regeneration is axonal growth from injured neurons and sprouting is axonal growth from uninjured neurons. Both may contribute to functional recovery. DLK and LZK are mammalian homologues of invertebrate DLK that has been shown to play important roles in axon regeneration in C. elegans and Drosophila. The role of mammalian DLK and LZK in spinal cord repair in not known. In the process of studying DLK (MAP3K12) and LZK (MAP3K13) in axonal repair after spinal cord injury, we have identified a critical role for LZK in astrocytic scarring. This result corroborates with published literature on Stat3 and Pten to illustrate an emerging theme that signaling pathways regulating axonal repair may also regulate astrocyte response to injury. In this proposal, we will comprehensively investigate the neuron and astrocyte specific roles of LZK and DLK in the multicellular response to spinal cord injury using an array of inducible loss and gain of function mouse genetic lines. Downstream effectors of DLK and LZK will be identified through transcriptomic analyses in neurons and astrocytes. Functional synergies or redundancies will be tested between DLK and LZK, and the interaction with other signaling pathways including Stat3 and Pten will be tested as well. Together, these studies will determine the contribution of DLK and LZK in axonal repair and astrocyte response after spinal cord injury, which may pave the way for therapeutic development targeting these molecules to promote repair and recovery after spinal cord injury.