Yimin Zou - US grants

DESCRIPTION (provided by applicant): The nervous system is composed of an enormous number of neurons, precisely connected into an intricate network, subserving the anatomical basis for all behaviors. Abnormal development of neural circuitry may cause various neurological and mental disorders. The long-term objective is to understand the molecular and cellular mechanisms of axon guidance during the wiring of the functional nervous system. The discovery of the proposed research will also provide clues for axonal regeneration studies following injury in the adult central nervous system. The mechanisms of axon wiring along the anterior-posterior axis of the central nervous system (the connections between the brain and the spinal cord) have remained a long-standing mystery. Wnt-Frizzled signaling was recently shown required for the A-P guidance of spinal cord commissural axons. However, the growth cone signaling mechanisms that mediate responsiveness to Wnt proteins are unknown. Three pathways are currently known to mediate Wnt actions: the canonical beta-catenin pathway, the planar cell polarity (PCP) pathway and the Ca++/PKC pathway. This grant proposes to test the role of the PCP pathway (Aim1), the PKC pathway (Aim2) and the canonical pathway and potentially novel pathways (Aim3) in axon guidance using commissural axons as a model system. This grant will also explore the possibility that more than one pathway might be involved in different aspects of guidance events, advancing the understanding cellular mechanisms of axon guidance. The finding that Wnt proteins can act as axon guidance cues provides more opportunity for understanding brain wiring. The proposed studies will provide foundation for future studies on the mechanisms how sensory pathways are guided to project anteriorly (from the spinal cord to the brain) and motor pathways to project posteriorly (from the brain to the spinal cord) and may also address the biological role of Wnt gradients in patterning neuronal networks in the central nervous system.

DESCRIPTION (provided by applicant): The nervous system is made of a large number of neurons, precisely connected by axons. This complex axonal network is important to all behavioral functions and is established largely during embryogenesis under the control of a variety of guidance molecules. Abnormal development may lead to errors in neuronal connections, causing various neurological and mental disorders. The long-term objective is to understand the growth cone signaling mechanisms controlling directional growth of axons in building neural circuits. Understanding molecular mechanisms underlying axon pathfinding may provide crucial tools for axonal regeneration in the adult central nervous system following injury. A merging theme in axon guidance is that multiple signaling pathways are frequently found to function in one single neuron, raising a fundamental question of how the signaling pathways are integrated in the same growth cone, the cellular apparatus located at the tip of the axon responsible for sensing guidance cues and growth cone steering. Two major chemorepellent pathways, the Semaphorin/Neuropilin pathway and the Slit/Robo pathway, were discovered separately. Preliminary studies suggest an unexpected crosstalk between the two signaling pathways. The goal of the proposed research is to explore the mechanism of the crosstalk between these two major chemorepellent pathways and the biological significance of growth cone signaling convergence. The proposed study will shed light on the organization of axon guidance signaling pathways and further the understanding of molecular mechanisms of nervous system development .

DESCRIPTION (provided by applicant): Neuronal synapses play pivotal roles in neural circuit functions. Abnormal synapse formation leads to numerous developmental diseases of the nervous system with cognitive and behavioral disabilities, including Down Syndrome, Angelman Syndrome, Fragile X Syndrome and Autism Spectrum Disorders. The mechanisms of how synapses form during development are poorly known. Understanding the basic mechanisms of synapse formation will be essential to understand the underpinnings of many birth defects in the nervous system for diagnosis and treatment. Several promising candidates for inducing synapse formation, such as the neuroligins and neurexins, are found not essential for synapse formation. Therefore, the question of synaptogenesis remains unsolved. Wnts have been shown to be able to regulate synapse formation in several embryonic neurons in hippocampus, cerebellum, spinal cord and the neuromuscular junction. Our preliminary results show that Wnt/planar cell polarity (PCP) signaling is required for excitatory synapse formation in dissociated hippocampal neuronal culture and in the neuromuscular junction in vivo. We propose to test the hypothesis that Wnt/PCP signaling is the central pathway, which directly assembles pre- and post-synaptic structures. PUBLIC HEALTH RELEVANCE: Abnormal synapse formation leads to numerous developmental diseases of the nervous system with cognitive and behavioral disabilities, including Down Syndrome, Angelman Syndrome, Fragile X Syndrome and Autism Spectrum Disorders. Understanding the basic mechanisms of synapse formation will be essential to understand the underpinnings of many birth defects in the nervous system for diagnosis and treatment. Several promising candidates for inducing synapse formation, such as the neuroligins and neurexins, are not essential for synapse formation. Therefore, the question of synaptogenesis remains unsolved. We propose to characterize the role and mechanisms of Wnt/PCP signaling in formation of excitatory synapses.

DESCRIPTION (provided by applicant): Axons in the central nervous system do not regenerate readily after traumatic injury because of the highly inhibitory environment of the adult central nervous system especially following injury. In addition, adult axons generally lack intrinsic growth potential and therefore cannot sustain the initial sprouting observed in some of the injured axons. Furthermore, injured axons often retract further back from where they are lesioned over time, making repair even more challenging. Therefore, combinatorial approaches will be necessary for successful anatomical regeneration and functional recovery. Many axon guidance molecules important in development are still present in the adult central nervous system after the nervous system. Still others are found re- induced after injury. Wnts are guidance cues that control pathfinding of a number of axons along the rostral-caudal axis of the developing spinal cord. Wnts attract ascending axons via their seven-span transmembrane receptors, Frizzleds, and repel others via a different transmembrane receptor, Ryk, in the spinal cord. Our studies showed that the re-induced Wnt signaling system regulates the growth cone of axons in the injured adult central nervous system. Ryk-mediated Wnt repulsion causes the well-known retraction/die back of injured corticospinal tract axons and limits the regenerative potential of proprioceptive sensory axons even after conditioning lesion of their peripheral branches, whereby crushing the peripheral branch enhances the growth state of the central branch of sensory axons. This grant tests whether more effective regeneration can occur by combinatorial approaches. We will test whether combining Wnt signaling manipulation and PTEN deletion can further enhance the regenerative potential of conditioning lesion.

? DESCRIPTION (provided by applicant): The mechanisms of how growth cones integrate simultaneous guidance instructions or how they modify responsiveness to sequential cues along their trajectories have not been well understood. Our preliminary study revealed a novel gene-expression-based switch mechanism. Our previous work showed that planar cell polarity (PCP) signaling is essential for their proper anterior-posterior (A-P) guidance after midline crossing. We now found that Sonic Hedgehog (Shh) induces a subset of PCP genes in commissural neurons during midline crossing. We propose that this novel regulatory loop between Shh and PCP signaling may also be essential in many other cases of axon guidance or in developmental processes other than axon guidance. In this exploratory R21 grant, we propose to understand the mechanisms of how Shh-Smo signaling activates the PCP gene expression.

Spinal cord injury (SCI) can result in long-term loss of sensory and motor functions due to axon damage, gliosis, inflammation, and demyelination. Transected axons fail to regenerate in the adult mammalian CNS due to 1) lack of intrinsic regrowth ability, 2) inhibitory extrinsic cues including myelin associated proteins and chondroitin sulfate proteoglycans enriched in the glial scar as well as axon guidance molecules and 3) a physical gap of neural tissue for axon growth. Astrocytes constitute one of the principal components of the glial scar. In response to SCI, astrocytes become reactivated and start proliferation. How astrocyte reactivation is initiated and what cellular signaling pathways are involved in astrocyte polarization is largely unknown. Therefore, we hypothesize that Wnt/PCP signaling may be an important regulator of polarization of the reactive astrocytes after spinal cord injury, which may be important for glial scar formation and glial bridge formation.

Project Summary Severed CNS axons fail to regenerate largely because of the reduced intrinsic growth capacity of adult neurons and poor environment for axon extension. The Co-I's Lab finds that the Wnt family molecules and their receptors are upregulated after spinal cord injury (SCI) and mediates regrowth Among adult failure of injured fiber tracts. several Wnt receptors, Ryk is crucial for mediating repulsive axon growth during development and in CNS after injury.Chondroitin sulfate proteoglycans (CSPGs) generated by glial scars strongly suppress axon extension and are major extrinsic molecular targets for treating CNS injury. The PI's group designed small peptides to block functions of CSPG receptors LAR and PTP? by targeting their critical activity domains and demonstrated their high efficiency for promoting axon growth. Blocking each of the two receptors with 3 combined peptides promotes robust regeneration of injured CNS axons. We hypothesize that inhibiting both Wnt and CSPG signals represents a dual approach of enhancing neuronal growth capacity and reducing environmental inhibitory influence at the lesion site. We propose to stimulate robust axon regrowth in adult rodents with transection or contusion SCI by inhibiting Ryk and LAR/PTP? with genetic and pharmacological approaches available in our labs. In Aim 1, we will study synergistic actions of transgenically deleting Ryk plus each of LAR/PTP? receptors on promoting axon regeneration and recovery in mice with SCI. We will determine whether deleting Ryk plus LAR or Ryk plus PTP? receptors acts synergistically to stimulate axon growth and enhance neuronal plasticity in double knockout mice after SCI. In Aim 2, we will determine whether blocking each of Ryk, LAR and PTP? receptors with antibody or selective antagonists pharmacologically promotes axon regeneration and recovery in adult rats with SCI. We will compare effectiveness of the treatments that target individual receptors in promoting regrowth of multiple descending tracts and recovery of locomotor functions after SCI. In Aim 3, we will study whether combination therapies that block two or three receptors yield better axon regrowth and functional recovery in rats with transection or contusion SCI, aiming to identify the optimal therapy for mammals with SCI. Based on the promising results from our pilot studies, we predict that our combined strategies will promote dramatic regeneration of injured axon tracts and recovery of locomotion function in vivo. Our novel strategy of administering deliverable compounds post-injury may facilitate development of a practical combinatorial therapy for CNS lesions.

The glutamatergic synapses are the main excitatory synapses in the brain. Abnormal synapse formation and plasticity are responsible for numerous diseases, such as intellectual disability, autism, neuropsychiatric and degenerative disorders. The signaling mechanisms controlling their assembly and plasticity have not been fully understood. Our preliminary studies lead to the surprising finding that components of the highly conserved cell polarity signaling pathways are important regulators. We found that components of both planar cell polarity (PCP) and apical-basal polarity (A-BP) pathways are localized in developing excitatory synapses and interact with multiple key presynaptic and postsynaptic proteins. In this proposal, we propose to test several hypotheses on how these key cell polarity components regulate glutamatergic synapse formation and plasticity.

Autism and epilepsy have strong genetic components and significant comorbidity. Many mutations associated with both autism and epilepsy occur in the genes important for synapse formation, function and plasticity, suggesting overlapping molecular as well as circuit mechanisms. Understanding the defects of synapse development caused by these mutations will provide important insights to pathogenesis of both diseases and tools for therapeutic intervention. We found that components of planar cell polarity (PCP) pathway are localized in developing excitatory synapses and interact with multiple key presynaptic and postsynaptic proteins. Several point mutations in PRICKE1 and PRICKLE2 have been identified in patients with myoclonus epilepsy. Using CRISPR-Cas9-mediated gene editing, we generated mice that carry these human mutations. In this proposal, we will test whether these mice can serve as animal models to study how abnormal development can lead to these disorders.

The goal of this new Gordon Research Conference (GRC) series, entitled ?Central Nervous System Injury and Repair?, together with its accompanying Gordon Research Seminar (GRS), is to provide a forum for the exchange of the most exciting and cutting edge scientific data and novel ideas in the rapidly advancing fields of injury, regeneration, and repair of the central nervous system, with particular emphasis on spinal cord injury. Spinal cord injury is a major medical challenge since it leaves millions of people worldwide paralyzed and with loss of sensation, cardiorespiratory, bladder and sexual dysfunction as well as pain for their lifetime. Despite many years of efforts, an effective treatment or cure is still lacking. This is in part due to the complexity of this disorder where in addition to the damage of axons, spinal cord injury leads to profound changes of the cellular and molecular environment, involving neurons, glia, immune cells, etc. An emerging consensus is that combinatorial approaches targeting multiple cellular and molecular aspects of the injury response will be necessary to treat spinal cord injury and induce functional recovery. As spinal cord is an essential part of the central nervous system and spinal cord injury is a challenging disorder, we think a current focus on the spinal cord will help facilitate communications and advance the cutting-edge science. Because many issues of the central nervous system are shared between the spinal cord and the brain, the current focus on spinal cord injury will benefit the research of central nervous system injury and repair in general. The 2021 Central Nervous System Injury and Repair GRC will feature the most exciting work on the molecular, cellular, and systems-level changes that affect the injured spinal cord and neural repair. There will be 9 sessions of oral presentations ranging in topics from basic science to clinical translation. Each presentation will be accompanied by ample time to query results and discuss the implications for the larger health community, including patients, healthcare professionals, and policy-makers. This interactive forum can collectively accelerate discovery and translation of treatments that will lead to functional recovery. We further aim to have attendance, including oral and poster presenters, from basic and physician scientists and from industry, as well as a breath of diversity across gender, geographical locations and age groups to enable a broad communication of the scientific content. We will be aggressively sensitive to the goal of diversifying attendance by disseminating conference information to organizations and institutions with under-represented individuals. Perhaps one of the most exciting developments will be the introduction of a GRS that accompanies the GRC, which will be essential for the future success of the field. The GRS is a symposium held immediately preceding the GRC that provides a platform for young scientists to discuss their research and respond to real-time queries from other young scientists. The goal of the GRS is two-fold: to fuel scientific communication amongst peers and to provide career mentoring for young scientists by their more senior colleagues.