Jonah R. Chan - US grants

DESCRIPTION (provided by applicant): A number of genes have been discovered that are responsible for the demyelinating diseases of the peripheral nervous system. Exploring the mechanisms of demyelination caused by alterations in these genes also illuminates the molecular mechanisms of myelination. For example, it appears that mutations in the peripheral myelin protein 22 (PMP22) gene differentially alter the interactions of the PMP22 protein and in turn alter its trafficking pattern. An offshoot of these studies was the discovery that brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3), besides being survival factors for sensory neurons, are also mediators of the myelination process. In this proposed study the identity and location of the neurotrophin receptors that are responsible for the actions of BDNF and NT3 will be determined. The experiments involve the use of function blocking antibodies or receptor antibodies and of neurotrophin mutants that bind selectively to either the trk receptors or to the p75NTR. Location of the receptors will probed by in situ hybridization. Two complementary myelination systems will be used, one in cocultures of sensory neurons and Schwann cells and the other in the developing sciatic nerve. The expression of the receptors identified in cocultures will be explored in the in vivo system and in the developing sciatic nerve of viable animals deficient in appropriate neurotrophin receptors or neurotrophins. Besides identifying one more ligand-receptor system involved in myelination the data may also aid in considering the therapeutic application of neurotrophins in the peripheral demyelinating disease.

DESCRIPTION (provided by applicant): Cell polarity is critical for various cellular processes including establishing the antero-posterior axis, generating distinct membrane specializations (apical and basal polarity), as well as asymmetric cell division and axon specification. Essentially, cell polarity plays fundamental roles in helping to organize and integrate complex molecular signals in order for cells to make decisions concerning fate, orientation, differentiation, and interaction. In the nervous system, neurons and glia share a mutual dependence in establishing a functional relationship, and none is more evident than the process by which glia form myelin around axons. The formation of myelin is an exquisite example of cell-cell interaction, which consists of the polarized or unidirectional wrapping of multiple layers of membrane concentrically around an axon initiated at the site of the axon-glial interface. While myelination is a highly polarized process, the involvement of cell polarity in its formation remain largely uncharacterized. We have recently identified a novel role for the Par (partitioning defective) polarity complex in the initiation of myelination. This polarity complex localizes asymmetrically in myelin- forming cells at the SC-axon interface, and disruption of Par localization, dramatically inhibits myelination without affecting cell division, migration, or even axonal alignment. The central hypothesis of this proposal is that axonal signals facilitate the breaking of symmetry in the SC and initiate myelination by coordinating cytoskeletal dynamics/rearrangement and gene expression. Our recent findings provide us with a rare opportunity to characterize the presence of this polarized molecular scaffold at the SC-axon interface that leads to the unidirectional activation of myelination. A clear understanding of the molecular and cellular events that pave the way for the myelin-forming cell is vital in advancing therapies for demyelinating diseases such as Multiple Sclerosis, the peripheral neuropathies, and even nerve injury.

SUMMARY Damage to myelin from diseases such as multiple sclerosis (MS) results in the disruption of the nerve signal, damage to the axon, and finally degeneration. To date, there are no therapies for repair or remyelination in MS and this fact alone illustrates the greatest hope and unmet need for MS patients. Functional screening for small molecules or biologicals that promote remyelination represents a major hurdle to the identification and development of rational therapeutics for MS. Recently we implemented a novel functional screen using fabricated micropillar arrays to identify anti-muscarinic compounds that greatly enhance oligodendrocyte remyelination (Mei et al., 2014). As many of the promising compounds identified in our initial screen activated or antagonized G-protein coupled receptor (GPCR) targets, in this proposal, we focus screening efforts on GPCR small molecule libraries to identify/confirm/validate receptor targets that either inhibit or promote myelination. We believe that GPCRs represent targetable receptors and pathways for the development of small molecule therapeutics for MS. In this proposal we will: 1. Perform high-throughput screening of GPCR small molecule libraries to identify agonists and antagonists that promote myelination. 2. Identify, confirm and validate novel receptors and pathways responsible for the regulation of oligodendrocyte differentiation and myelination. 3. Investigate the therapeutic implications of activating or blocking specific receptors during development and after demyelination. Overall, we believe that our proposal will not only impart a valuable technical approach but more importantly our data identifies two specific GPCRs, the muscarinic receptor 1 (M1R) that inhibits (Gq) and the kappa opioid receptor (KOR) that promotes (Gi/Go) differentiation and myelination of oligodendrocytes both during development and after demyelination.

PROJECT SUMMARY An underappreciated aspect of myelination is the avoidance of selecting non-axonal targets. Oligodendrocytes (OLs) select axons while avoiding neuronal somata, dendrites and processes of other glial cells. How is this specificity accomplished? While OLs are capable of myelinating permissive structures (artificial fibers and beads) in the absence of molecular cues, structurally permissive neuronal somata and dendrites remain unmyelinated. These observations suggest that myelin substrate selection is not cell-intrinsically limited to physiologically relevant geometries. OL cell processes are likely sensitive to cell-extrinsic cues that ensure the selection of axons with high fidelity. Utilizing a novel purified spinal cord neuron-OL myelinating coculture system we find that disruption of dynamic neuron-OL signaling by chemical crosslinking results in aberrant myelination of the somatodendritic compartment of neurons. In this proposal, we hypothesize that inhibitory somatodendritic cues act as repulsive membrane signals that are necessary and sufficient to prevent non-axonal myelination. In this proposal we will: 1. Perform next-generation sequencing and candidate profiling of membrane proteins expressed exclusively in purified spinal cord neurons. 2. Identify, confirm and validate expression and localization of repulsive membrane proteins enriched in the somatodendritic compartment. 3. Investigate the necessity and sufficiency of the repulsive signal(s) to prevent aberrant myelination and identify receptor(s) on OPCs that mediate the inhibition. Our preliminary data identifies the Junctional Adhesion Molecule 2 (JAM2), expressed on the somata and dendrites of spinal cord neurons, as a repulsive signal that inhibits aberrant oligodendrocyte myelination. Taken together, we propose a model in which broadly indiscriminate myelination is tailored by inhibitory signaling to meet local myelination requirements.

PROJECT SUMMARY Oligodendrocyte precursor cells continually produce myelinating oligodendrocytes in the adult brain throughout life. Active myelination of adult brain circuits has been shown to be important for some forms of learning, and recent work from our groups has shown that this a crucial process in memory storage and retrieval. However, while previous work has provided essential insight into the regulation of myelin plasticity in the adult brain, it is not clear how this process impacts the dynamic nature of neural encoding within memory circuits. Myelination increases conduction velocity across individual axons, however how this translates to computations at the level of neural circuits and their subsequent behavioral outputs is poorly understood. Thus, a considerable gap exists between those findings related to axonal myelination in the adult and those that describe the neural coding dynamics that underlie memory encoding, consolidation and recall. In this proposal we aim to bridge this gap by 1) elucidating the cell types in the mouse medial prefrontal cortex that become myelinated after a learning experience, 2) determine how active myelination of cortical circuits impacts the cellular codes that support memory, 3) how activity-dependent myelination modulates the synchronization and interregional communication between the medial prefrontal cortex, amygdala and hippocampus during fear memory recall and 4) the temporal dynamics of oligodendrocyte precursor proliferation and differentiation in vivo after memory encoding. These studies will provide the first ever evidence for bidirectional interaction between new myelin formation and active memory encoding ensembles and will elucidate fundamental mechanisms of glial signaling during learning and memory.

This proposal focuses on addressing one of the most fundamental questions regarding OL biology: What axonal cues in the CNS microenvironment control OL differentiation and myelination? While it is still yet unclear whether the spatial and temporal patterns of myelination are dependent on inductive or inhibitory cues (or both), we know that exclusively axons ? but not all axons ? are myelinated by OLs in parallel with neuronal circuit maturation. This suggests that axon-derived signals must be involved in coordinating this process. In this proposal, we have identified a novel axon-derived peptide class, namely dynorphins that promote OL differentiation and myelination. Neuropeptides, have several characteristics that make them an ideal axonal signal to regulate myelination. They are stored in dense core vesicles and released only in response to high levels of neuronal activity, a phenomenon that might signal a form of maturation that qualifies an axon for myelination. Neuropeptides bind to G-protein coupled receptors and have slow-acting effects that may include altering gene expression, providing a mechanism through which they might alter cellular fate. In this proposal we will investigate: 1. Whether OLs and their precursors are influenced by the neuropeptide class, dynorphin, 2. Whether dynorphins are released in response to neuronal activity to regulate myelination and 3. Whether dynorphins influence myelination globally or is restricted only to dynorphin expressing axons. Recent studies demonstrate that biophysical properties of fiber diameter, inhibitory molecules and neuronal activity may all affect OL precursor cell (OPC) proliferation, differentiation, and the selection of axons for myelination (Gibson et al., 2014; Hines et al., 2015; Mensch et al., 2015; Redmond et al., 2016; Mitew et al., 2018; Mayoral et al., 2018). Here, we provide the molecular mechanism and downstream signaling pathways for a specific subset of neurons that may underlie activity dependent differentiation and myelination. Our preliminary data place us in a unique position to determine whether dynorphins are a neuropeptide class that represents an axonal cue to control OL differentiation and myelination. We believe that these findings should impart valuable insight in providing a framework for identifying additional neuropeptides and transmitters that may influence oligodendroglial lineage cells, as well as for profiling inhibitory and inductive cues for myelination.