Shai Shaham - US grants

DESCRIPTION (provided by applicant): Programmed cell death (PCD) is an essential part of development, and is used, among other things, to sculpt organs and to discard cells whose functions are no longer required. Although global regulators of PCD have been well characterized, little is known about cell-specific signals that regulate PCD during development. Our long-term goal is to identify and characterize cell-specific developmental signals that impinge on the global PCD machinery to regulate a cell's survival or demise. C. elegans is an excellent organism for studying regulation of PCD. Studies in this organism identified genes (ced-3, ced-4, and ced-9) that define a core pathway controlling virtually all PCD. Homologs of these genes (caspase proteases, Apaf- 1, and Bcl-2 family members, respectively) can function in a similar pathway in mammals. To understand how this pathway is regulated in specific C. elegans cells we will pursue two main objectives: 1) We will perform genetic screens to identify genes regulating PCD of the male-specific CEM neurons and PCD of the embryonic tail-spike cells. While CEM PCD is likely to be controlled by cell-autonomous processes, PCD of the tail-spike cells may be controlled by a signaling cascade that induces transcription of the core PCD gene ced-3. We have already identified two genes, cdr-1 and cdr-2, that prevent CEM death in hermaphrodites. Their roles in CEM PCD will be explored. 2) We will define the role of the CED-9-interacting protein CIP-l in PCD in the male tail. CIP- 1, a BH3 -domain protein, promotes PCD when over-expressed and is exclusively transcribed in the developing male tail. We will generate mutations in the gene, define residues essential for its function, and study its intracellular localization. Misregulation of PCD is a hallmark of many disease states in humans. For example, excess PCD is observed in several neurodegenerative diseases, and lack of PCD is seen in cancer. The remarkable conservation of the PCD machinery between C. elegans and humans strongly suggests that our studies will yield insight into how human PCD is regulated as well.

DESCRIPTION (provided by applicant): Programmed cell death (PCD) is important for animal development and understanding this process may provide insight into human ailments such as cancer and neurodegenerative disease. Although much is known about global control of apoptotic cell death, relatively little is known about cell-specific signals that regulate PCD during development. Our long term goals are to understand the molecular features that distinguish cells destined to live from those destined to die;to understand how temporal control of cell death is achieved;and to determine whether caspase-dependent cell death is the only mode of cell death used during animal development. C. elegans is an excellent organism in which to study the control of PCD. Dying cells in developing animals are easily visualized, and genetic and molecular studies of PCD genes are generally facile. Most PCD in C. elegans is regulated by an evolutionarily conserved molecular pathway. Specifically, CED-3/caspase promotes PCD following activation by CED-4/Apaf-1. This process is kept in check by CED-9/Bcl-2, which inhibits CED-4, and by EGL-1, a BH3 domain-containing inhibitor of CED-9. It is widely believed that control of cell-specific death and its onset are regulated by controlling EGL-1 activity. However, we demonstrated two instances in which this paradigm does not hold up. First, in the tail-spike cell, CED-9 and EGL-1 play only a minor role in the control of cell death. In this cell, onset of death appears to be controlled by transcriptional induction of the ced-3 gene using the PAL-1 transcription factor, the C. elegans homolog of the vertebrate tumor-suppressor gene Cdx2. Second, in the linker cell, a program independent of all known cell death genes previously described in C. elegans, regulates cell death. Remarkably, this non-apoptotic program is morphologically similar to forms of vertebrate cell death that have not been studied in detail, suggesting that the process is likely to be conserved. Here we plan to pursue two specific aims: (1) We will use genetic and molecular approaches to understand how ced-3 transcription is controlled in the tail-spike cell to promote cell death. (2) We will use genetic strategies to uncover the mechanism by which the novel cell death pathway we discovered executes linker cell death. Given the morphological and molecular similarities between cell death programs in C. elegans and vertebrates, our studies are likely to reveal basic principles of cell death regulation common to these organisms. Public health: Inappropriate cell death underlies a host of human illnesses including neurodegenerative diseases, and lack of cell death is a major contributor to tumor development. Our studies will shed light on basic mechanisms regulating cell death, and should define proteins that may be used as therapeutic targets.

DESCRIPTION (provided by applicant): Glia constitute a large fraction of cells in the vertebrate nervous system and surround neuronal receptive endings to form isolated compartments. Most excitatory synapses in the cerebellum and hippocampus are glia-ensheathed. Likewise, glia surround sensory-neuron receptive endings and neuromuscular junctions. While glial compartments influence sensory responses and synaptic transmission and plasticity, the development and functions of glial compartments are only incompletely understood. Our long-term goals are to establish robust in vivo settings for studying glia-neuron interactions, and to use these settings to fully understand glial compartment development and function. Sensory organs are highly suitable systems in which to study these basic principles. They exhibit simple architecture and are of critical importance to animal and human behavior, as they are the portals through which information is introduced into the nervous system. Sensory organs consist of two cell types: sensory neurons or neuron-like cells and glia or glia-like cells, which are required for neuron function. A major advantage of sensory organs is their remarkable similarity across a wide range of organisms. This allows studies in one system to reveal principles conserved in others. The amphid sensory organ of C. elegans is a prototypical sensory organ and is the most studied sensory organ in C. elegans; however, how its glial compartment is formed has not been investigated. Since we initiated our studies of glial signaling compartments, we have characterized a novel mode of dendrite growth that properly positions glial compartments, have characterized neuronal proteins required for sensory receptive ending structure, revealed a key role for glia in regulating sensory neuron receptive ending shape, demonstrated glial developmental plasticity and uncovered its mechanism, and characterized signaling between sensory neurons and their ensheathing glia to promote glial compartment formation. Here we propose to build on our progress to understand both glial and neuronal mechanisms controlling glial compartment size and shape. We will (1) elucidate the mechanism of action of the LIT-1 NEMO-like kinase in glial compartment size control; (2) study the role of the retromer component SNX-1 in glial compartment morphogenesis; and (3) identify neuronal signals required to localize LIT-1 to the glial compartment surface. Together, these studies should provide insight into the dynamic cell interaction and cell shape changes required to form a glial compartment around sensory neuron receptive endings.

DESCRIPTION (provided by applicant): Our long-term aim is to understand how sensory organs form. Sensory structures allow organisms to assess their environment, and sensory organ defects in humans lead to perceptive and psychological deficits. Lumen formation plays key roles in sensory organ development. Often, a tubular channel formed by glia or epithelia is penetrated by neuronal endings exposed to the environment to form the organ. How this coordinated tubulogenesis occurs is not known. Biological tubes allow liquid and gas flow in plants and animals. In humans, tubes are essential components of most organs. Defects in tube formation and maintenance, or ectopic formation of tubes, underlie numerous human disease states, including vascular aneurysms, intestinal herniations (diverticulosis), and congenital defects of the vasculature, intestine, kidney, and lungs. Excessive vascular tubulogenesis can support malignant growth. Little is known about the molecular mechanisms regulating tube lumen formation and size. The C. elegans amphid sensory organ is an excellent structure in which to study sense organ development. It is composed of 12 neurons with well-defined sensory roles, and two tube-forming glial cells that ensheath the neurons. Amphid architecture is remarkably similar to some sensory organ structures in Drosophila and mammals. Cells of the amphid are easily labeled with fluorescent reporter proteins, and their development can be examined in real time. Furthermore, genetics and molecular biology are generally facile in C. elegans, allowing for discovery of conserved molecular players regulating channel formation. We showed that a key regulator of C. elegans lumen formation is encoded by the daf-6 gene. DAF-6 protein is related to vertebrate and Drosophila Patched, and is expressed in all C. elegans tube classes, localizing to luminal surfaces. We have also shown that mutations in the wrt-6 hedgehog-like gene result in amphid defects similar to those of daf-6. The existence of a Hedgehog-Patched module has not been demonstrated in C. elegans and our results suggest that wrt-6 and daf-6 may indeed be such a module. Finally, we showed that LIT-1/Nlk kinase is a target of DAF-6 signaling, and is, thus, the first described target of Patched-related signaling in C. elegans. Here we propose three aims: (1) We will study the role of wrt-6 in lumen formation. (2) We will study lit-1 function in lumen formation. (3) We will clone previously isolated daf-6 suppressor mutations to reveal additional components of the new signaling pathway we discovered. Because Patched and related proteins are important in many aspects of human development and tumor formation, studies of the new signaling pathway we identified will help understand the functions of these key developmental proteins. In addition, our studies will provide insight into tubulogenesis, a process important for the formation of all organs, defective in congenital diseases, and hyperactivated in cancer. Public Health Relevance: Because Patched and related proteins are important in many aspects of human development and tumor formation, studies of the new signaling pathway we identified will help understand the functions of these key developmental proteins. In addition, our studies will provide insight into tubulogenesis, a process important for the formation of all organs, defective in congenital diseases, and hyperactivated in cancer.

DESCRIPTION (provided by applicant): Our long-term aim is to understand the mechanism by which neuronal receptive-ending shape is altered by experience. In the nervous system, cell shape is malleable. Neuronal receptive endings, such as dendritic spines and sensory protrusions, are structurally remodelled by experience, and an emerging hypothesis in cellular neuroscience is that these shape changes accommodate and define changes in neuron output. Alterations in receptive-ending structures may, therefore, underlie nervous system plasticity, and may contribute to complex cognitive capacities including learning and memory. How receptive-ending structures acquire and change shape is not well understood; however, it has been assumed that a direct response of postsynaptic neurons to presynaptic activity accounts for most aspects of the phenomenon. Here we challenge this view, suggesting that glial cells associated with receptive endings play major roles in determining receptive-ending shape, and therefore function, together with presynaptic cues. Glia are the most abundant cell type in the human brain, and glia contribute extensively to nervous system disease. However, the roles played by glia in the nervous system remain largely mysterious. Several observations suggest that glia could influence the shapes of neuronal receptive-endings: they are in the right place at the right time, they can sense the postsynaptic milieu, their shapes correlate dynamically with neuronal receptive-ending cell shapes, and mutations in some glial proteins affect receptive ending shape. We previously demonstrated that the nematode C. elegans offers a unique arena in which to explore glial functions in the nervous system, allowing in vivo studies of glial function to be adresed in ways curently not posible in vertebrate settings or even in Drosophila. We propose to use the powerful methods of genetic analysis in C. elegans to uncover 1) the molecular mechanisms by which glia affect neuronal shape, and 2) how remodeling afects neurons function and animal behavior. In the longer term, we plan to explore conservation of the pathways we identify beyond C. elegans. Achieving a comprehensive understanding of the mechanisms that endow nervous systems with anatomic and behavioural plasticity is of paramount importance in understanding the brain. Such an understanding should, eventually, allow us to tackle human disorders, including learning disabilities and autism, which may result from alterations in synaptic function and plasticity.

DESCRIPTION (provided by applicant): Our long-term goal is to understand a novel nonapoptotic developmental cell death program we discovered, and its relationship to polyglutamine-induced neurodegenerative disease. Cell death is a major cell fate during metazoan development. Apoptosis, an extensively studied cell death process, requires caspase proteases and is accompanied by a stereotypical morphological signature. Surprisingly, mice lacking apoptotic effectors survive to adulthood, raising the possibility that non- apoptotic cell death may play key roles in animal development. Thus, a major unsolved question is whether alternative developmental cell death pathways exist, and if so, what molecular mechanisms govern their execution. We recently discovered that the death of the C. elegans male-specific linker cell (LC) is not apoptotic. LC death has neither apoptotic, nor autophagic or necrotic morphological features. Instead, the dying LC displays pronounced indentation (crenellation) of the nuclear envelope, uncondensed chromatin, and swelling of the endoplasmic reticulum and mitochondria. Importantly, LC death is independent of CED-3 caspase, all other caspases, and all other known C. elegans apoptotic proteins, including CED-4/Apaf-1, CED-9/Bcl-2 family, and EGL-1 and CED-13 BH3-domain-only proteins. These exciting findings demonstrate that LC death must occur through a novel mechanism. From a genome-wide RNAi screen for genes promoting LC death we identified two genes, pqn-41, a gene of previously unknown function, and let-70, encoding an E2 ubiquitin conjugating enzyme. Loss of either gene blocks nuclear crenellation, but not organelle swelling. The pqn-41C transcript as well as let-70 promote LC death, and are expressed in the LC only as cell death is initiated. LC death displays striking ultrastructural similarities to nonapoptotic developmental cell death in the vertebrate nervous system. Several observations also suggest similarities to polyQ-induced neurodegeneration. Like polyQ proteins, PQN-41C is highly glutamine rich, and forms coiled-coil secondary structures. Like polyQ proteins, PQN-41C aggregates in cells. Furthermore, ultrastructural studies of polyQ disease tissue reveal changes similar to those seen during LC death, including nuclear envelope crenellation and organelle swelling. Here we propose to understand the mechanisms of action of pqn-41 and let- 70, and related mammalian proteins, and to pursue a small molecule screen to identify inhibitors of LC death. Together, these studies should not only inform us about the process of LC death, but may yield important clues and reagents as to the etiology of polyQ-dependent neurodegenerative disease. PUBLIC HEALTH RELEVANCE: Our long-term goal is to understand how cell death takes place during normal animal development and disease. We recently uncovered a novel cell death process in the nematode C. elegans with similarities to normal and disease-related cell death in humans. Here we aim to understand the details of this novel killing mechanism, and to study its relatedness to human disease. Our studies may, therefore, provide in-roads towards understanding and, perhaps, treatment of degenerative human diseases.

DESCRIPTION (provided by applicant): Our long-term goal is to understand a novel nonapoptotic developmental cell death program we discovered, and its relationship to polyglutamine-induced neurodegenerative disease. Cell death is a major cell fate during metazoan development. Apoptosis, an extensively studied cell death process, requires caspase proteases and is accompanied by a stereotypical morphological signature. Surprisingly, mice lacking apoptotic effectors survive to adulthood, raising the possibility that non- apoptotic cell death may play key roles in animal development. Thus, a major unsolved question is whether alternative developmental cell death pathways exist, and if so, what molecular mechanisms govern their execution. We recently discovered that the death of the C. elegans male-specific linker cell (LC) is not apoptotic. Instead, the dying LC displays pronounced indentation (crenellation) of the nuclear envelope, uncondensed chromatin, and swelling of the endoplasmic reticulum and mitochondria. Importantly, LC death is independent of CED-3 caspase, all other caspases, and all other known C. elegans apoptotic proteins, including CED-4/Apaf-1, CED-9/Bcl-2 family, and EGL-1 and CED-13 BH3-domain-only proteins. These exciting findings demonstrate that LC death must occur through a novel mechanism. From a genome-wide RNAi screen for genes promoting LC death we identified several genes required for LC death, including one encoding a protein rich in glutamines. LC death displays striking ultrastructural similarities to nonapoptotic developmental cell death in the vertebrate nervous system and several observations also suggest similarities to polyglutamine-induced neurodegeneration. Here we propose to (1) to study aspects of PQN-41 function and determine functions of interacting proteins; (2) characterize new LC death genes identified from a genetic screen; and (3) understand the control of LC death. Given the similarities between LC death and vertebrate's cell death, our results may contribute towards an understanding of cell death processes in human development and disease.

Our long-term goal is to understand glial control of neuron receptive-ending shape and function. Neurons detect external stimuli through specialized dendritic receptive endings. Receptive-endings are malleable and regulate neuronal output. For example, neurons receive information at dendritic spines. Remodeling of spine shape occurs in development and is effected by experience. Perturbations in spine shape are associated with neurological disorders, suggesting that spine morphogenesis may play key roles in nervous system function. Like spines, receptive endings of sensory neurons are remodeled in development and by experience. Photoreceptor cell outer segments, for example, are turned over and rebuilt daily. Perturbation of sensory receptive-ending shape leads to sensory deficits and is a common pathology in sensory diseases. Despite clear clinical importance, the questions of how sensory cell shapes are regulated, how shape affects function, and how glia-neuron interactions control sensory neuron receptive-ending shape, have not been extensively addressed. The nematode C. elegans is an excellent organism in which to study glia-neuron interactions controlling sensory receptive-ending morphogenesis and plasticity. Sensory organ anatomy, physiology, and molecular biology are conserved from C. elegans to humans, making the nematode an exciting arena for revealing general principles of sensory organ development and function. The C. elegans AFD neuron mediates temperature sensation and cultivation-temperature memory. We showed that AFD receptive- ending shape is dynamically controlled and uncovered a novel signaling pathway guiding these changes. Shape changes require the receptor guanylyl cyclase GCY-8, controlling cGMP levels in AFD. High cGMP blocks receptive-ending extension, and this is overcome by overexpression of the actin regulator WASP-1. Loss of the glial transporter KCC-3, which specifically surrounds AFD, also blocks microvilli growth, by removing Cl- ions from an extracellular microdomain around AFD. Cl- ions function as novel direct inhibitors of GCY-8 cyclase activity. We further found that glia engulf and take up AFD neuron receptive-ending fragments, and that engulfment is required for AFD neuron function. Our results reveal glia-neuron interaction pathways determining neuron receptive-ending morphology, components of which are conserved in mammals. We propose three aims: (1) We will determine how glia form a unique microdomain around AFD neurons that is distinct from domains around other neurons. (2) We will study the role of glial FIG-1/thrombospondin in engulfment of AFD receptive endings and AFD neuron shape. (3) We will study the role of the phosphatidylserine receptor PSR-1 in engulfment of AFD endings and shape.

Our long-term goal is to understand how glia contribute to nervous system development, function, and information processing. Glia constitute a large fraction of cells in the vertebrate nervous system and surround neuronal receptive endings to form isolated compartments. Most excitatory synapses are glia-ensheathed, as are sensory-neuron receptive endings and neuromuscular junctions. Major gaps remain in our understanding of glia. While developmental specification of some glia has been explored, programs governing astrocyte or sensory organ glia differentiation are not clear. How glia form and regulate compartments around synapses and other neuronal receptive endings is also not understood. Glia have been proposed to regulate neuronal activity, yet the effector mechanisms are not fully explored. Finally, neuron structural and functional plasticity may, in part, be under glial control, yet the details are not at hand. Thus, much remains to be learned about glial functions and their underlying molecular programs. In many animals, neurons are born in excess, and the final neuronal complement is determined in part by glial and other secreted cues controlling cell death. Glial manipulation, thus, often leads to neuronal demise. A long-standing goal has been to identify in vivo settings for studying glia-neuron interactions that bypass the neuron-survival problem. We have taken a major step towards this goal by pioneering the nematode C. elegans as a facile and relevant system for studying glia and their nervous system contributions. We showed that C. elegans possess glia, and that these ensheath sensory-neuron receptive endings, highly resembling glial structures found in vertebrate sense organs, as well as envelop the CNS, wrapping around defined synapses. Like vertebrate astrocytes, these latter glia tile, subsuming specific CNS domains, express transcription factors promoting gliogenesis in vertebrates, and express ion and neurotransmitter transporters, channels, and neurotransmitter receptors. The development of these glia bears uncanny similarities to the radial glia-to-astrocyte developmental transition in vertebrate brain development. Importantly, in C. elegans, neuron survival does not require glia, but glia manipulation results in major deficits in neuron shape and function. C. elegans therefore offers a unique in vivo arena to study glia and their effects on the nervous system. Here we aim to investigate three interrelated aspects of glia-neuron biology. (1) We will determine how astrocytic glia develop and regulate synaptic function. (2) We will determine glia guided brain assembly. (3) We will study a new cell death program resembling glia-dependent neurodegeneration. In addressing these questions, we challenge the view that only neurons underlie the phenomena under study, and posit that glia are integral regulators.

Our long-term goal is to understand the molecular basis of a novel morphologically-conserved non-apoptotic developmental cell-death program we uncovered, and to determine its roles in mammalian development and disease. Programmed cell death is a major cell fate. Apoptosis, an extensively studied cell death process, requires caspase proteases and is accompanied by chromatin compaction and cytoplasmic shrinkage. Surprisingly, mice lacking apoptotic effectors survive to adulthood. These observations suggest that non- apoptotic cell death may play key roles in animal development. Although genes promoting necrotic cell death have been described, these are not required for development. Thus, whether alternative developmental cell death pathways exist, and if so, what molecular mechanisms govern their execution, is a major outstanding question. Our studies of the C. elegans linker cell provide direct evidence that caspase-independent non- apoptotic cell death pathways operate during animal development. Linker cell death occurs in the absence of C. elegans caspases, and other apoptosis genes are also not required, nor are genes implicated in autophagy or necrosis. The morphology of a dying linker cell is characterized by lack of chromatin condensation, a crenellated nucleus, and swelling of cytoplasmic organelles. Remarkably, cell death with similar features (linker cell-type death, LCD) also occurs in vertebrates, and is characteristic of neuronal degeneration in polyglutamine diseases. We recently described a pathway governing C. elegans LCD. This is the first such framework for a non-apoptotic developmental cell-death program. LCD is controlled by Wnt signals that function in parallel with a developmental-timing and a MAPKK pathway to control non-canonical activity of HSF-1, a conserved heat-shock transcription factor. let-70/Ube2D2, encoding a conserved E2 ubiquitin- conjugating enzyme, is a key target of HSF-1. The E3 components CUL-3/cullin, RBX-1, BTBD-2, and EBAX-1 function with LET-70/UBE2D2 for LCD. Our recent evidence suggests that histone methylation may be a target of this pathway, likely resulting in genome-wide chromatin opening, allowing nuclease access and DNA degradation. LCD pathway components promote vertebrate cell-degenerative processes. pqn-41, a glutamine- rich protein, is reminiscent of polyQ proteins causing neurodegeneration. and tir-1/Sarm and BTBD-2 promote distal axon degeneration following axotomy, supporting conserved cell dismantling roles. We recently showed that treatment of mammalian cells with the kinase inhibitor staurosporin (STS) causes LCD like death. Here we will build on these studies to uncover LCD pathway targets, and study relevance to mammals. We will: (1) Investigate the role of SAMS-4, a BTBD-2 target, and NUC-1, a DNaseII enzyme, in LCD, and test an hypothesized pathway for these in chromatin modification and DNA degradation. (2) Identify EBAX-1 target genes and assess roles in LCD control. (3) Characterize STS-induced death in mammalian cells, define conservation with C. elegans LCD, and identify relevant genes.