Shanthini Sockanathan, Ph.D. - US grants

The coordinated generation of specialized groups of spinal motor neurons during development of the central nervous system is essential for the assembly of functional neural circuits. Analysis to date hasfocused on the role of the signaling molecule shh in motor neuron specification however increasing evidencesuggests that other inductive molecules may play pivotal roles in their development. The dynamicspatiotemporal expression of RALDH2, the major synthetic enzyme for retinoic acid suggests that retinoidsignaling is required at defined stages in the sequential development of motor neurons. However, loss offunction studies have not been informative due to the early lethality of RALDH2 null embryos. This proposalaims to test the hypothesis that retinoids are required at specific stages of motor neuron development usinggenetic strategies in the mouse to bypass the early lethality of RALDH2 mutants. Tissue specific knockouts of RALDH2 will be constructed and analyzed to first assess the contribution of paraxial mesoderm derived retinoids to motor neuron generation and column induction and second, to determine if motor neuron derived retinoid signaling is necessary for motor column, motor division and motor pool determination. Finally, four prospective target genes for RALDH2 have been isolated using differential screening approaches and experiments outlined here will focus on functional analyses to determine the potential role of two of these genes in motor neuron specification. Taken together, these experiments aim to define the contribution of retinoic acid signaling to motor neuron development with the ultimate aim of assembling a molecular pathway of retinoid dependent events required for their specification during embryogenesis. Understanding the processes by which motor neuron specification occurs may provide insight into the basis of motor neuron degenerative diseases such as amyotrophic lateral sclerosis or the spinal muscular atrophies. This in turn may lead to the design of innovative treatments for these diseases in the future which may incorporate the use of stem cell technology.

DESCRIPTION (provided by applicant): The major goals of this proposal are to understand the molecular mechanisms of motor neuron differentiation and specification. Retinoid (RA) signals are necessary for synchronizing neurogenic and motor neuron specification pathways during spinal motor neuron differentiation and these events are mediated by the RA-responsive gene, GDE2. GDE2 encodes a six transmembrane protein with an extracellular glycerophosphodiester phosphodiesterase (GDPD) domain. GDPD activity is required for GDE2's ability to coordinate cell-cycle exit and motor neuron specification, revealing a novel link between GDPD metabolism and motor neuron differentiation. The distinctive topology of GDE2 where the GDPD domain is extracellular and the lack of functional precedent strongly predicts the discovery of new molecular networks involved in motor neuron differentiation. To identify such networks, unbiased screens were used to isolate proteins that interact with GDE2. Components of two different signaling pathways were identified. In this proposal, in vitro structure-function analyses will be combined with in vivo loss- and gain- of function studies to investigate how these signaling pathways integrate with GDE2 to promote motor neuron differentiation. While onset of GDE2 expression occurs in differentiating cells, GDE2 expression is maintained in terminally differentiated motor neurons;suggesting that GDE2 may have additional roles critical for later motor neuron function or survival. To define the function of GDE2 at different stages of motor neuron development, mouse genetics will be used to ablate GDE2 in differentiating and postmitotic motor neurons. Resultant embryos will be analyzed for defects in motor neuron differentiation, motor neuron specification, motor axon target recognition and motor neuron survival. These experiments will determine how two different signaling pathways synergize with GDPD- dependent modes of neuronal differentiation and further advance current understanding of the regulatory networks involved in cell differentiation. In addition, these studies will investigate potential roles for GDE2 in diverse cellular processes critical for neuronal function and survival.

DESCRIPTION (provided by applicant): The major goals of this proposal are to understand the molecular basis of neuronal and glial differentiation in the developing nervous system. Here, the mechanisms that control sequential motor neuron and oligodendrocyte differentiation from a single progenitor domain (pMN) within the spinal cord will be investigated. GDE2, a six-transmembrane (6-TM) protein that contains an extracellular glycerophosphodiester phosphodiesterase (GDPD) domain is necessary and sufficient to induce motor neuron differentiation through GDPD activity. In this proposal, biochemical and in vivo approaches will be used to define the mechanism of GDE2 GDPD activity and identify potential physiological substrates of 6- TM GDPD enzymatic function. Strikingly, a second 6-TM GDE protein shows complementary expression to GDE2 that is coincident with oligodendrocyte generation. In vivo and in vitro approaches will be taken to test the function of this second GDPD regulatory system in oligodendrocyte specification, differentiation and maturation. Genetic ablation of GDE2 results in increased numbers of oligodendrocytes in the gliogenic period, suggesting that GDE2 might prevent the premature initiation of oligodendrocyte differentiation. This hypothesis will be tested using a combination of biochemical, genetic and functional approaches. Taken together, these studies will provide mechanistic insight into novel GDPD-dependent regulatory systems that control motor neuron and oligodendrocyte differentiation, and determine if they intersect to regulate the temporal control of neurogenesis and gliogenesis in the spinal cord.

? DESCRIPTION (provided by applicant): This proposal imagines a paradigm shift for understanding human Alzheimer's disease that is based on a newly discovered, druggable enzyme. Our current understanding of AD proposes a central role for the accumulation of amyloid A? (1), but there is great need for advances in understanding mechanisms that cause increases of A? in sporadic AD. Moreover, there are many aspects of the pathology of AD that cannot be simply understood as a consequence of A? accumulation, and suggest other molecular mechanisms contribute to pathogenesis. There is also a great need for diagnostics that can be rationally linked to pathogenesis. Most of all there is a need for effective therapeutics. These challenges are well known to the field. We see a unique opportunity to advance AD research that builds on the discovery of a novel enzyme family that controls several of the most important signalling pathways in brain development. GDEs catalyze cleavage of the phosphodiester bond that links a class of extracellular proteins to the cell surface (2). These GPI-linked proteins act as activators or inhibitors of Notch, sonic hedgehog, fibroblast growth factor, Wnt, ephrin (EphA5), ciliary neurotrophic factor receptor (CNTF), glial derived neurotrophic factor, and contactins. The role of GDEs in neurodegeneration was made serendipitously with the discovery that conditional deletion of GDE in adult brain results in profound, age-dependent neurodegenerative changes that include many of the hallmarks of human neurodegenerative disease. We hypothesize that loss of GDE function contributes to human neurodegenerative disease. The approach exploits the fortunate consequence of GDE activity, which is to shed substrate proteins into the extracellular compartment and CSF. This creates biomarkers of GDE activity. As proof of concept, we have determined that prion protein is a substrate of GDE. Prion protein is present in the CSF at levels that are reduced in human AD subjects in parallel with cognitive decline(3), and recent studies implicate prion protein in pathological reduction of synaptic strength and enhanced A? generation in AD(4, 5). We will expand this precedent using non-biased methods to identify GDE substrates in CSF and brain of normal and diseased humans. These biomarkers will identify specific signalling pathways consequent to GDE function that are consistently disrupted in AD. Where successful, this approach will provide mechanism-linked biomarkers of disease that can be combined with modulators of the GDE pathway as new mechanism-based diagnostics and therapeutics for AD.

This proposal examines a novel, neuronal signaling pathway implicated in amyloid precursor protein (APP) processing. A? amyloid accumulates in brain as a defining aspect of Alzheimer's disease and is generated in the ?amyloidogenic pathway? via the sequential processing of APP by ? secretase (BACE1) and ?-secretase. APP is also processed by ?-secretase ADAM10 (a disintegrin and metalloprotease 10), which is termed the ?non-amyloidogenic? pathway since it prevents A? generation. While a great deal is known about BACE1 and ?-secretase, less is known about mechanisms that control ADAM10 processing of APP. We have discovered a signaling pathway that controls ADAM10 processing of APP in neurons, and in preliminary studies demonstrate its dysregulation in human Alzheimer's disease brain. This pathway includes plasma membrane proteins GDE2 (glycerophosphodiester phosphodiesterase 2), an enzyme that catalyzes cleavage of GPI (glycosylphosphatidylinositol) linked proteins; RECK (reversion-inducing cysteine-rich protein with Kazal motif, which is an inhibitor of ADAM10 and a GPI linked protein that is ?shed? from the plasma membrane by GDE2; and ADAM10. In support of the importance of this signaling pathway, Gde2 KO mice exhibit a pronounced reduction in ?-secretase cleavage products and a dramatic shift toward amyloidogenic processing of APP. Moreover, levels of membrane RECK are increased in Gde2 KO brain, and expression of membrane-tethered RECK is sufficient to inhibit endogenous ?-secretase activity in neurons. Preliminary studies examining human postmortem brain document biochemical and histochemical evidence of GDE2 loss of function and robust elevation of membrane RECK in Alzheimer's patients. We hypothesize that GDE2 control of RECK surface expression regulates ADAM10 ?-secretase activity and that disruption of this pathway shifts equilibrium of APP processing towards the amyloidogenic pathway. Aim 1 will define the mechanism of RECK inhibition of ADAM10 ?-secretase activity and will determine contributions to amyloidogenic APP processing. Aim 2 will test the hypothesis that GDE2 regulation of surface RECK expression is a critical determinant of ADAM10 ?- secretase function. Aim 3 will determine physiological contributions of GDE2 and RECK to the onset and progression of A? pathogenesis in new knock-in mouse models of amyloidosis. Our studies will determine if the GDE2-RECK pathway is a critical determinant of ADAM10 ?-secretase activity; outcomes could provide new molecular perspective into mechanisms of A? pathogenesis in human Alzheimer's disease.

Project Summary Glia have emerged as important contributors to spinal motor neuron survival; however, the mechanisms by which they promote motor neuron viability are not well understood. This proposal will examine a novel pathway in oligodendrocytes that regulates the survival of spinal motor neurons in the adult nervous system. This pathway includes the six-transmembrane enzyme GDE2 (glycerophosphodiester phosphodiesterase 2; GDPD5), which cleaves the GPI (glycosylphosphatidylinositol)-anchor that links proteins to the cell-membrane, and the GPI- linked heparan sulfate proteoglycan Glypican (GPC) 6. In support of this pathway, Gde2 knockout (KO) mice show progressive motor neuron degeneration that culminates in motor neuron loss and motor deficits. Timed and cell-specific genetic ablation of GDE2 associates motor neuron pathologies with postnatal GDE2 function and additionally, pinpoints GDE2 cellular requirement for motor neuron survival to oligodendrocytes. Unbiased screens to isolate GPI-anchored GDE2 substrates combined with biochemical evaluation in an independent model of motor neuron degeneration, identify GPC6 as a potential mediator of GDE2 function in motor neuron survival. GDE2 releases GPC6 through GPI-anchor cleavage, and cell-based assays identify a second GDE2- dependent mechanism of GPC6 release by extracellular vesicles (EVs). Further, conditioned medium containing cleaved and EV GPC6 rescues motor neuron loss of Gde2 KO organotypic spinal cord cultures. These observations suggest the hypothesis that GDE2 constitutes a physiological pathway in oligodendrocytes that promotes spinal motor neuron survival, and that GDE2 neuroprotective function is mediated through release and delivery of GPC6. Proposed experiments will test this hypothesis in two specific aims. Aim 1 will define properties and specificity of EV and cleaved GPC6 to motor neuron survival in context of the GDE2 pathway, and explore the contribution of released GPC6 to motor neuron survival in physiological and pathological settings. Aim 2 will determine consequences of GDE2 loss in oligodendrocytes on GPC6 release, and will examine physiological requirement of GPC6 expression in oligodendrocytes to motor neuron survival. Outcomes from the proposed studies are expected to identify the GDE2-GPC6 pathway as essential for motor neuron viability, and to provide new mechanistic insight into oligodendrocyte contributions to neuronal survival.

Two causal mechanisms of neurodegeneration that are found in Alzheimer's disease (AD) and AD-related diseases such as amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD) are re-initiation of the mitotic cell-cycle in neurons, and impaired nucleocytoplasmic trafficking resulting from disruptions in the nuclear envelope (NE) and nuclear pore complex (NPC). Deeper insight into how these abnormalities arise would clarify approaches to design effective treatments for these diseases; however, very little is known about the initiating mechanisms involved. This application will test the hypothesis that neuronal quiescence is maintained throughout life via active mechanisms that inhibit the mitotic cell-cycle and that re-initiation of the cell-cycle leads to NE/NPC disassembly, a normal occurrence in mitotic cells. This hypothesis is based on our study of the six-transmembrane enzyme GDE2 (Glycerophosphodiester phosphodiesterase 2; GDPD5), which cleaves the GPI-(Glycosylphosphatidylinositol)-anchor that tethers some proteins to the plasma membrane. GDE2 is a potent inhibitor of the mitotic cell-cycle and induces the differentiation of mitotic progenitors into post-mitotic neurons in the developing nervous system. We discovered that in adult mice lacking GDE2 (Gde2 KO), cortical neurons show evidence of cell-cycle re-entry, suggesting that GDE2 is required to preserve neurons in a quiescent state. Strikingly, Gde2 KO neurons that have re-entered the cell-cycle show abnormal organization of the NE, aberrant distribution of NPC proteins and impaired nucleocytoplasmic transport, raising the possibility that cell-cycle re-initiation and NE/NPC breakdown are linked. Consistent with this idea, genetic reduction of cyclin D, a critical regulator of the G1/S transition, suppresses nucleocytoplasmic transport-dependent neurodegeneration in a Drosophila model of c9ORF72 ALS-FTD. Notably, Gde2 KO mice display age-progressive neurodegeneration and GDE2 distribution and function is disrupted in AD patient neurons. These observations suggest that maintenance of neuronal quiescence is an active process and that failure of this process re-initiates the cell-cycle, triggers NE/NPC breakdown and induces neurodegeneration. Aim 1 will determine if GDE2 encodes a new pathway that maintains neuronal quiescence and will determine if neuronal cell-cycle re-entry signals NE/NPC breakdown in neurons. Preliminary RNAseq, analysis of Wnt-reporter mice in Gde2 KOs, and genetic studies in Drosophila identify aberrant activation of canonical Wnt signaling as a candidate pathway that induces neuronal cell-cycle re-entry, NE/NPC breakdown and neurodegeneration. Aim 2 will utilize mouse and Drosophila models to test this hypothesis. Studies in Aim 3 will determine links between GDE2 dysfunction, neuronal cell-cycle re-entry and NE/NPC breakdown in disease using Drosophila models of AD and ADRD, human postmortem tissue and iPS human neurons. These studies will provide new molecular insight into cross-disease triggers of neurodegeneration important in human AD and ADRDs.