Matthew N. Rasband - US grants

DESCRIPTION (provided by applicant): Ion channel clustering in myelinated axons is essential for proper nervous system function. Ion channels are clustered at nodes of Ranvier through neuron-glia interactions. However, the mechanisms responsible for channel clustering remain poorly understood. Recent studies suggest that multiple mechanisms may contribute to node formation. Among these, the axonal submembranous cytoskeleton comprised of ankyrins and spectrins has been proposed to be key components. In particular, nodes of Ranvier themselves are enriched with ankyrinG (ankG) and ßIV spectrin; ankG is thought to bind directly to the Na+ and K+ channels, and then link to the actin cytoskeleton through ßIV spectrin. At paranodes, both ankyrinB and ßII spectrin are clustered, although their functions at paranodes remain unknown. Paranodes are also thought to function as a paranodal diffusion barrier and a second mechanism to mediate ion channel clustering at nodes, although the mechanisms responsible for this barrier function remain unknown. Furthermore, a major impediment to elucidating the function of paranodes is the relatively few proteins that have been identified at this site. In this proposal we will seek to determine the function of the nodal and paranodal cytoskeletons in node of Ranvier assembly and maintenance. We will do this using three new mouse models that utilize Cre-Lox technology to control the temporal and spatial (cell-type specific) expression of ankG, ankB, and ßII spectrin. We will silence expression of these proteins in peripheral sensory neurons, in retinal ganglion cells, and in myelinating glia during both development and in adults. Finally, we will use proteomic methods to identify new paranodal proteins, and then seek to determine their functions.

? DESCRIPTION (provided by applicant): This application requests continued support for the fifth renewal of the NIGMS supported training grant for graduate training in neuroscience at Baylor College of Medicine. The goal of the Neuroscience Graduate Program is to provide a broad-based interdisciplinary training program in the neurosciences that prepares students to be the future leaders in neuroscience. Our training program spans the diversity of research topics found in modern neuroscience, including, but not limited to, molecular and cellular neuroscience, systems neuroscience, and cognitive and computational neuroscience. The training plan is designed to develop competency in both practical and theoretical aspects of neuroscience including modern laboratory techniques, genetics, cell biology, neurophysiology, biochemistry, and computational methods. The program includes 49 faculty from a variety of departments at Baylor College of Medicine. These faculty have diverse research interests that span the basic, translational, and clinical neurosciences. There are currently 57 students enrolled in the program, and 11 (19%) of these students are underrepresented minorities or have disabilities. We accept ~10 students into the program each year, with 6 supported by the NIGMS training grant, and the remainder through institutional funds provided to the Department of Neuroscience by Baylor College of Medicine. The Neuroscience Graduate Program has a rigorous and broad curriculum in the neurosciences that is supplemented with courses in many areas to prepare students for the diverse research topics they pursue. These courses include topics such as modern methods in neuroscience, developmental neurobiology, systems neuroscience, and the neurobiology of disease. The strength of our Neuroscience Graduate Program is reflected in the excellent publication rates of our graduates (3.2 papers/student with an average of 1.7 as first author) and the outstanding and committed training faculty. We believe that the Graduate Program in Neurosciences at Baylor College of Medicine is an exceptional place to pursue training in the Neurosciences and that this application appropriately documents our commitment and tradition of excellence. We therefore seek support to continue our efforts to train the next generation of neuroscientists who will make discoveries that will impact human health and well-being. We request funds to sustain our current program with 8 training grant positions per year.

DESCRIPTION (provided by applicant): Rapid and efficient propagation of action potentials in the mammalian nervous system requires both myelination and the high-density clustering of voltage-gated ion channels at gaps in the myelin sheath called nodes of Ranvier. Although many demyelinating diseases and injuries cause disruption of nodes and nervous system dysfunction, the mechanisms that are responsible for ion channel clustering at central nervous system (CNS) nodes of Ranvier remain unknown. We propose that three distinct cellular and molecular interactions contribute to CNS node formation and maintenance: 1) interactions between axonal cell adhesion molecules and a unique CNS nodal extracellular matrix, 2) interactions between axons and myelinating glia at paranodal junctions set up a membrane protein diffusion barrier to restrict the lateral mobility of nodal proteins, and 3) interactions between nodal membrane proteins and nodal cytoskeletal scaffolds maintain high density clusters of ion channels. Thus, multiple, overlapping mechanisms may exist in the CNS to facilitate ion channel clustering at nodes of Ranvier. In this project we will undertake both cell biological and genetic methods to determine the mechanisms underlying CNS node of Ranvier formation. We will focus on the extrinsic, glial-derived interactions that are necessary for CNS node formation. In the first aim we will elucidate the molecular interactions between nodal cell adhesion molecules and CNS nodal ECM proteins. We will determine if soluble ECM proteins are sufficient to induce clustering of nodal proteins in purified neuronal cultures. In the second aim we will perform genetic analyses of single, double, and triple knockout mice lacking extracellular matrix molecules, paranodal junctions, and/or cytoskeletal interactions to uncover the existence of, and requirement for, each overlapping mechanism. PUBLIC HEALTH RELEVANCE: Disruption of nodes of Ranvier or their molecular composition is one consequence of demyelination and contributes to the pathophysiology of many diseases and injuries including multiple sclerosis and spinal cord injury. Thus, any therapeutic effort aimed at treating these diseases or reversing their devastating effects will require a detailed understanding of the mechanisms responsible for node of Ranvier formation and maintenance. Nodes have been the focus of much interest not only because of their functional importance in both health and disease, but also because their assembly represents one of the best examples of the elaborate reciprocal interactions that must occur between neurons and glial cells.

? DESCRIPTION (provided by applicant): Ion channel clustering at nodes of Ranvier is an essential feature of myelinated axons. Nodal Na+ channel clusters confer several important functional advantages including decreased energy and space requirements, and increased action potential conduction velocity. We recently showed that multiple, overlapping mechanisms contribute to CNS node of Ranvier formation. These mechanisms include both intrinsic (neuronal) and extrinsic (glial mechanisms). Extrinsic mechanisms include interactions between axonal cell adhesion molecules and glia-derived extracellular matrix molecules, as well as an axoglial junction-dependent diffusion barrier that restricts membrane proteins to nodes. Intrinsic mechanisms depend on cytoskeletal and scaffolding proteins. The redundancy of nodal Na+ channel clustering mechanisms makes genetic mouse models the only tractable approach to discover how CNS nodes are formed. Despite these advances, the molecular details for how these mechanisms work remain poorly understood and are even controversial. Here, we will use newly developed genetic mouse models to elucidate the molecular mechanisms responsible for node of Ranvier formation and maintenance. We propose to determine the roles of NF186 and the nodal ECM in node of Ranvier assembly and maintenance. We propose to determine how spectrins function to link the nodal Na+ channel protein complex to the underlying actin cytoskeleton. And finally, we propose to determine how paranodes function as lateral diffusion barriers. The experiments proposed here continue and extend our previous studies that identified multiple, overlapping mechanisms for CNS node of Ranvier formation.

? DESCRIPTION (provided by applicant): The newborn brain is particularly sensitive to hypoxic injury (HI). Preterm HI manifests itself as periventricular leukomalacia (PVL), while in full-term infants HI presents as hypoxic ischemic encephalopathy (HIE). In newborns with moderate-severe forms of either PVL or HIE, 60-75% develop life-long neurological disabilities, resulting from extensive white matter injury (WMI), due to the loss of myelinating oligodendrocytes (OLs). This loss of OLs, coupled with their failure to regenerate, leads to impaired neuronal function, which clinically manifests as cerebral palsy (CP). In this proposal we will attack the critical problem of remyelination after HI using two distinct approaches: the differentiation of OLPs and the maintenance of axonal integrity. Our studies have identified four compounds that act on distinct pathways that contribute to the suppression of remyelination, which we will test in the neonatal brain during- and after- HI. One feature of OLPs populating white matter lesions is elevated levels of Wnt signaling, which functions to suppress regenerative myelination after WMI. Therefore, inhibition of Wnt signaling in OLPs represents a therapeutic strategy for stimulating remyelination after HI. Recently we identified Daam2 as a key proximal modulator of Wnt signaling in the developing CNS that functions through the PIP5K-PIP2 signaling axis. Leveraging this knowledge from development, we found two compounds that inhibit PIP5K activity (e.g. Sp-8-pCPT-cAMP and UNC3230) stimulate remyelination after WMI after acute hypoxia. Here we will determine whether these compounds function similarly in the neonatal brain after HI and ischemia, and whether the Daam2-PIP5K axis is expressed in OLPs in human HIE/PVL lesions. Axon integrity also plays a central role in myelination. Recently, we found that disruption of the axon initial segment (AIS) in cortical neurons blocks their eventual myelination due to loss of axonal identity. Moreover, we found that the AIS is disrupted after ischemic injury in the adult brain. Thus, we propose HI- induced loss of the AIS inhibits myelination, whereas preservation of the AIS may promote myelination after HI. Ischemic injury activates the calcium dependent protease, calpain, which proteolyzes essential AIS scaffolding proteins, resulting in the loss of axonal integrity. Calpain inhibitors (e.g. MDL28170) preserve the AIS after ischemia both in vitro and in vivo. Therefore, we will determine if maintenance of the AIS through inhibition of calpain stimulates remyelination and recovery after HI, and whether these components of the AIS are dysregulated in human HIE/PVL lesions.

Project Summary: Ion channel clustering in myelinated axons is essential for proper nervous system function. Ion channels are clustered at nodes of Ranvier through neuron-glia interactions. However, the mechanisms responsible for channel clustering and neuron-glia interactions remain poorly understood. Our work shows that multiple glia-dependent mechanisms contribute to node formation. Furthermore, these mechanisms converge on axonal and glial ankyrin and spectrin cytoskeletons. In this project, we will determine the importance of nodal spectrin cytoskeletons by analyzing conditional knockout mice lacking ?I and ?IV spectrins, singly and in combination. We also discovered that AnkyrinR (AnkR) can function as secondary Na+ channel clustering mechanism. However, the primary functions of AnkR in the nervous system are unknown. We generated a conditional allele for AnkR and will use this in loss-of-function studies to determine the role of AnkR in various cells throughout the nervous system. We performed differential proteomics on WT and AnkR-deficient brains together with immunoprecipitation to identify AnkR interacting proteins. We will investigate these interactions to further define the functions of AnkR in the nervous system. Finally, in a discovery aim, we will use a newly generated mouse (Nfasc-BioID) to perform in vivo proximity biotinylation at nodes and paranodes. We will then capture these proteins using streptavidin affinity purification, followed by mass spectrometry to identify biotinylated proteins. This will begin to uncover the node and paranode interactomes. We will validate and investigate these biotinylated proteins through localization, and gain and loss of function studies both in vitro and in vivo.

Project Summary This application requests continued support for the sixth renewal of the Neuroscience Graduate Program (NGP) training grant at Baylor College of Medicine. The goal of the NGP is to provide a broad- based interdisciplinary training program in the neurosciences that prepares students to be the future leaders in neuroscience. The NGP spans the diversity of research topics found in modern neuroscience, including, but not limited to, molecular and cellular neuroscience, systems neuroscience, and cognitive and computational neuroscience. The training plan is designed to develop core competencies in both practical and theoretical aspects of neuroscience including modern laboratory techniques, genetics, cell biology, neurophysiology, biochemistry, and computational methods. Furthermore, we aim to develop competencies in critical thinking, quantitative analysis, and effective oral and written communication. We also emphasize rigor, reproducibility, and ethics in our training plan. The program includes 60 training faculty from departments at Baylor College of Medicine. These faculty have diverse research interests that span the basic, translational, and clinical neurosciences. There are currently 65 students enrolled in the program. We aim to accept ~12 students into the NGP each year. We request support for 6 students, to be funded in their 1st year on the training grant, with the remainder of students supported through institutional funds provided to the NGP by the Baylor College of Medicine Graduate School of Biomedical Sciences. The NGP has a rigorous and broad curriculum in the neurosciences that is supplemented with courses in many areas to prepare students for the diverse research topics they pursue. These courses include topics such as modern methods in neuroscience, neurophysiology, systems neuroscience, computational neuroscience, and the neurobiology of disease. The strength of the NGP is reflected in the excellent publication rates of our graduates (4.4 papers/student with an average of 1.8 as first author) and the outstanding and committed training faculty. We believe that the NGP at Baylor College of Medicine is an exceptional place to pursue training in the Neurosciences and that this application appropriately documents our commitment and tradition of excellence. We therefore seek support to continue our efforts to train the next generation of neuroscientists who will make discoveries that will impact human health and well-being.

Na+ channels are highly clustered at the neuromuscular junction (NMJ) deep in the junctional folds below acetylcholine receptors (AchRs). Whereas AchRs respond to the release of acetylcholine from the motor neuron and are responsible for the initial membrane depolarization, the clustered Na+ channels are responsible for the muscle action potential. AchR clustering depends on a combination of agrin, neuregulin, and activity dependent mechanisms that have been described in great detail. However, little is known about the mechanisms responsible for NMJ Na+ channel clustering. Diseases including myotonia, periodic paralysis, and myasthenic syndrome all disrupt the NMJ. Na+ channel clustering occurs at two other locations in the nervous system including nodes of Ranvier and axon initial segments (AIS). Here, both cytoskeletal and extracellular interactions participate in channel clustering and the mechanisms have been described in detail. Remarkably, many of the same proteins involved in Na+ channel clustering at nodes and AIS are also found at the NMJ. By analogy to nodes and AIS, we propose that NMJ Na+ channel clustering depends on similar cytoskeletal and extracellular interactions. Aim 1 will consist of two parts designed to determine the cytoskeletal interactions important for NMJ Na+ channel clustering. First, we will conditionally knockout (specifically in skeletal muscle) the three Na+ channel-binding ankyrins (Ank1-3) singly and in combination. Second, we will conditionally knockout the 4 different spectrins known to be expressed in muscle and that are thought to link ankyrins (and Na+ channels) to the actin cytoskeleton. In both ankyrin and spectrin deficient mice we will evaluate muscle function and Na+ channel clustering. In Aim 2 we will identify the extracellular interactions that participate in NMJ Na+ channel clustering. First, we will generate muscle-specific knockouts of the cell adhesion molecule Nfasc given its location at the NMJ and its important role mediating extracellular interactions at the AIS and nodes. Second, since much less is known about the cell adhesion molecules and extracellular matrix molecules that may underlie NMJ extracellular interactions, we will use proximity biotinylation methods and proteomics to identify these proteins. We will then validate potential candidates for their localization to the NMJ, and using gain and loss of function strategies determine their functions. The aims proposed here will dramatically improve our understanding of the molecular mechanisms controlling Na+ channel clustering at the neuromuscular junction and may lead to important insights into the pathophysiology of neuromuscular diseases and neuropathies where NMJs degenerate or function is compromised.

New Abstract: The causes of complex neuropsychiatric disorders including schizophrenia, bipolar disorder, and autism remain poorly understood. Importantly, many neuropsychiatric disorders are highly heritable, indicating the underlying cause is genetic rather than environmental. Large genome-wide association studies (GWAS) have begun to reveal specific genes associated with these disorders. Among these, ANK3 (the gene coding for AnkyrinG (AnkG)) is potentially associated with bipolar disorder, schizophrenia, and autism. However, how ANK3 variants contribute to these disorders remains unknown. Importantly a loss-of-function ANK3 splice variant containing a novel exon (termed BDex) found only in oligodendrocytes may be protective against bipolar disease. We previously found AnkG in paranodes of myelinating oligodendrocytes, where it functions in the timely assembly of paranodal junctions during early development. However, the function of oligodendroglial AnkG in older mice remains unknown. We will determine the function of AnkG in oligodendrocytes, and more specifically the function of BDex. We will define the precise location of BDex-containing AnkG in oligodendrocytes. We will use loss-of-function and gain-of-function mouse models to investigate the role of oligodendroglial ANK3 variants in the brain. We will examine node and paranode structure, myelin, myelinated axon physiology, and behavior. Together, these studies will begin to uncover the role of oligodendroglial AnkG and BDex AnkG, and may reveal insights into the molecular pathophysiology of ANK3-associated neuropsychiatric disorders.

Project Summary Action potential initiation and propagation in myelinated axons requires high densities of ion channels clustered at axon initial segments (AIS), nodes of Ranvier, and a robust axonal cytoskeleton to help axons resist mechanicial injury. AIS also function to maintain neuronal polarity and regulate the distinction between axonal and somatodendritic domains. Unfortunately, disruption of these domains and the cytoskeleton during disease or after injury dramatically impairs nervous system function. Furthermore, the molecular mechanisms that control the assembly, function, and maintenance of AIS, nodes, and axonal cytoskeleton remain poorly understood. Since any therapeutic approach aimed at nervous system repair or regeneration must include the reassembly or preservation of axons, AIS and nodes of Ranvier, a detailed mechanistic understanding of their structure, mechanisms of assembly, and composition is urgently needed. To this end we developed proteomic approaches to perform a molecular dissection of AIS and nodes of Ranvier; these experiments will yield AIS and node 'interactomes.' To determine the functions of identified proteins we will perform rigorous gain and loss of function studies using modern molecular, imaging, genetic, and electrophysiological methods. Building on our previous research accomplishments and our discovery that mechanisms of node assembly converge on ankyrin and spectrin cytoskeletons, we will also determine the functions of these enigmatic, yet essential, cytoskeletal proteins using conditional knockout mouse models that we have developed. Together, we expect these studies to reveal key molecular mechanisms responsible for the assembly, maintenance, and function of axons. These discoveries may reveal targets and mechanisms that can be used for therapies to repair or preserve axon function.