1) The aims of this project is to develop proteomics methods to identify the substrates of protein phosphatase 1 (PP1) in neurons. The approaches will combine novel phospho-protein enrichment methods and state-of-the-art mass spectrometry. Using these methods, I will use brain tissue from wild-type and mutant mice lacking each of the three PP1 isoforms to identify substrates of each PP1 isoform. These proposed studies will establish general methods that can be used study protein phosphorylation, as well as reveal the functional differences between PP1 isoforms in neurons. I will then use the proteomics methods I have developed to study the roles of each PP1 isoforms in mice following behavioral tasks related learning and memory. 2) My previous research has focused on Cyclin-dependent kinase 5 (Cdk5). Using biochemical and molecular techniques, I have found phosphorylation-dependent proteolysis pathway in regulation of Cdk5 activity in neurons, and also revealed the functional role of Cdk5 in insulin secretion in pancreatic beta-cells. In the proposed project, I will change my focus from protein kinase to phosphatase, particularly PP1. In addition, I will develop and use various proteomics methods to identify new substrates for PP1 isoforms. 3) Together with my previous experience of kinases, the experience of studying phosphatases in this fellowship will complement my knowledge of protein phosphorylation mechanisms. I believe that the future studies will contribute to my broad and thorough understanding of phosphorylation-dependent signaling pathway. Moreover, the state-of-the-art proteomics methods will serve as a technical basis for my future research.

The actin cytoskeleton plays an important role in intracellular membrane trafficking. N-WASP, a regulator of actin assembly, is implicated in endocytosis and mediates actin-dependent rocketing of intracellular vesicles, but its precise cellular function is not well established. A recent study found that Abi1 binds to and regulates N-WASP activity in actin-based vesicular trafficking, as well as endocytosis of EGF receptor and transferrin receptor. Based on this and unpublished data that the SNARE domain of Abi1 is involved in its subcellular localization and that casein kinase 2, which is shown by others to phosphorylate N-WASP, was found to be an interactor of N-WASP by mass spectrometry studies, the following specific aims are proposed: 1) Test the hypothesis that the SH3 domain and other region(s) of Abi1 are required for full activity of Abi1 to stimulate N-WASP-dependent cellular processes. 2) Identification of the molecular determinants dictating Abi1 localization. 3) Test whether CK2 is involved in N-WASP-dependent cellular processes. This project will be a change of my research direction from membrane fusion in the early secretory pathway to the actin cytoskeleton and its regulation in intracellular trafficking. I will study a new aspect of membrane trafficking and learn and apply a wide range of biochemical, cell biological and molecular genetic approaches, which will significantly expand my knowledge and skills as a researcher in cell biology.

During development the basic wiring of the nervous system is established by connecting millions of neurons to their targets. In order to establish these connections, neurons send axons that navigate through the embryonic environment until they reach their targets and form synapses. The guidance of axons is directed by specific cues in the extracellular environment, and the past decade has seen the discovery of several families of guidance molecules and their receptors. Accurate guidance of an axonal tract requires the collaboration of multiple guidance cues. Thus, a central unanswered question in developmental neurobiology is how do multiple guidance cues spatially and temporally coordinate? With the help of the Career Development Award I would like to establish a rigorous research program that will address this question, using the guidance of sensory axons as a model system. Previous studies have demonstrated that Semaphorin-3A is an important guidance cue for sensory axons, and my work has shown that Plexin-A3 and -A4 serve as the primary signaling receptors for Semaphorin 3A. However, it is apparent that many additional cues collaborate with Semaphorin-3A to guide these neurons in an appropriate trajectory. Through the use of mouse genetics combined with in vivo and in vitro analyses I plan to identify cues that cooperate with Semaphorin-3A to guide sensory axons. Not only will these experiments increase our understanding of sensory system biology, more importantly these studies will uncover fundamental principles underlying the logic of axon guidance.

Cellular identities are defined by distinct gene expression programs that are set early during development and maintained via epigenetic mechanisms based on e.g. histone and DNA methylation as well as chromatin associated Trithorax and Polycomb group proteins. In mammalian pre-implantation embryos and primordial germ cells (PGCs), extensive changes in DNA methylation and histone modifications take place during the time of reacquisition of pluripotency. Importantly, members of the Polycomb Repressive Complex 1 (PRC1), implicated in homeotic gene silencing, X inactivation and stem cell renewal, are dynamically expressed during these developmental stages. Although epigenetic reprogramming events are thought to play a key role in establishing pluripotency, the precise underlying molecular mechanisms remain elusive. I propose to address the role of PRC1 in epigenetic reprogramming. First, I will use mice conditionally deficient for PRC1 components to examine PRC1’s regulatory role for higher order chromatin formation and directing cell fate in PGCs and pre-implantation embryos. Second, I will identify PRC1 target genes by genome transcription profiling using mutant mice. Third, I will address the significance of the PRC1 associated histone H2A lysine 119 mono-ubiquitination activity for epigenetic gene silencing in mammals. Through these projects, I will greatly extend my scientific horizon from my previous work on the regulation of chromosome segregation in fission yeast to the molecular mechanisms of epigenetic reprogramming in multi-cellular organisms. The proposed research will give me a thorough training in many aspects of epigenetic research and mouse molecular genetics.

Two distinct isoenzymes of adenosine deaminase ADA1 and ADA2 have been found in humans. Inherited mutations in ADA1 result in severe combined immunodeficiency (SCID). This observation has led to extensive studies of the structure and function of this enzyme that have revealed its important role in lymphocyte activation. In contrast, the physiological role of ADA2 is unknown. ADA2 is found in negligible quantities in serum and may be produced by monocytes/macrophages. ADA2 activity in serum is increased in various diseases in which monocyte/macrophage cells are activated. I have found that ADA2 is a heparin binding protein. This allowed me to obtain highly purified enzyme and to study its biochemistry. ADA2 was identified as a member of a new class of adenosine deaminase related growth factors (ADGF), which is present in almost all organisms from flies to humans. Biochemical data suggest that ADA2 may be active inside sites of inflammation during hypoxia and in areas of tumor growth where the adenosine concentration is significantly elevated and the extracellular pH is acidic. Our studies will explore the possibility that ADA2 is an immunomodulatory protein that may directly, or indirectly affect immune responses against intracellular pathogens or tumor cell proliferation. Our goal is to establish the physiological role of ADA2 in inflammation and tumor immunity. While this is a completely new direction for me given that my PhD work was on mechanisms of bacterial protein synthesis, the combination of my previous work on ADA2 biochemistry with the expertise of the Lauvau and Glaichenhaus laboratories in immunology will allow us to carry out the proposed experiments.

The canonical Wnt pathway plays an essential role in the establishment of colon cancer. The mutations implicated in tumor transformation lead to a stabilization of beta-catenin, inducing a constitutive activity of the pathway. TCF4 is a transcription factor activated downstream of Wnt. Beta-catenin/TCF4 has been shown by Clevers and colleagues to activate the genetic program in colorectal carcinoma cell lines, which inhibits differentiation and imposes a crypts progenitor phenotype to the cells. This genetic program is physiologically active in the gut controlling the switch between proliferation versus differentiation. Tcf4 knockout mice fail to maintain a proliferating stem cell compartment in the crypts. A micro-array performed in the lab has shown decreases in genes expressed in the crypts as well as in the mesenchymal cells surrounding the crypts. Thus the absence of crypt formation may induce secondary defects in the surrounding mesenchymal cells. The canonical Wnt pathway likely plays a crucial role in initiating epithelial-mesenchymal interactions. Since adenomas arise from the crypt-villus border similar interactions may be implicated in tumor transformation. In this proposal we aim to identify and functionally define genes expressed in the epithelial and mesenchymal cells involved in the formation of the crypt and stem cell niche. Subsequently we will investigate the relevance of these genes in adenoma formation. If successful this study may define novel therapeutic approaches in early intestinal neoplasia.

Some of the major respiratory viral pathogens (respiroviruses, rubulaviruses, morbillivirus and influenza virus) have in common the presence of glycoprotein spikes on their surface that mediate the first step of infection by binding to sialic acid-containing receptors on the surface of the host cell. In influenza viruses, haemagglutinin (HA) is the protein responsible for the adsorption of the virus to the host cell. The attachment of the virus takes place via multiple HA–receptor interactions acting cooperatively with effective binding resulting from an appropriate balance between binding affinity and receptor density. Studies in the literature have already demonstrated that multivalent compounds displaying multiple copies of terminal sialic acid groups bind influenza viruses with association constants that are several orders of magnitude those of monomeric sialic acid derivatives. However, little information is available on the influence of the macromolecular architecture of multivalent ligands on their ability to interact with influenza viruses. The aim of this project will be probing the virus/host cell interaction by testing well-defined and tailored multivalent glycoligands with different combinations of polydispersity, molecular weight, macromolecular shape and topology, as well as sialic acid content and distribution. Sialic acid-derived monomers will be synthesized that are suitable for radical polymerization. From these monomers, narrow polydispersity glycopolymers with a pre-determined molecular weight and shape will be prepared via living radical polymerization and their binding affinity and kinetics towards different influenza virus strains will be studied.

The molecular mechanisms that control CNS synapse formation and elimination are poorly understood. The Barres lab has identified an astrocyte-secreted protein, thrombospondin (TSP), which is sufficient to induce formation of structural synapses in vitro. They have subsequently shown that TSP induces the expression of the complement protein C1q in neurons. C1q is an immune system protein normally involved in phagocytic events, and is highly localized to synapses in the developing but not the adult brain. This raises the question of whether C1q may be involved in synapse elimination events, with astrocytic secretion of TSP controlling the timing of this elimination. The purpose of this proposal is to test the hypothesis that C1q helps to control the location of synaptogenesis in the developing brain through synapse elimination events, and that its re-expression in adult brain may play a role in synaptic loss in Alzheimer’s disease (AD). These studies have potential to shed new light on how synapses are eliminated during development, and how glial cells participate in this process through secreting factors that influence neuronal gene expression. In my previous research I characterized the mechanism of inhibitory neurotransmitter release during cerebral ischaemia using electrophysiological methods. The current research project will extend my knowledge into developmental neurobiology, as well as expanding my repertoire of techniques to include time-lapse imaging and cell culture. The ability to combine all of these techniques will provide me with a powerful means to investigate the influence of neuron-glial interactions in synapse formation, function, and plasticity.

Restoration of sight in a blind person imposes great clinical and scientific challenges. Despite intensive efforts, restoration of truly functional vision using neuroprostheses has not been achieved. I suggest here that a major reason for this failure is that the brain in the blind undergoes profound plastic changes and we do not know enough about how to communicate with this altered cortex to generate meaningful visual perception. The challenge is learning enough about these plastic changes to modify them with appropriate inputs in order to make the brain receptive to visual information once again. My previous work has included studies on brain plasticity in the blind. This proposal focuses on integrating this work with sensory substitution approaches, clinical ophthalmologic experience, and neuroprosthetic engineering training that are new for me. The work is truly multidisciplinary, bridging the Boston Retinal Implant Project and Harvard’s Center for Non-invasive Brain Stimulation. Specifically, I propose that sensory substitution devices (SSD), can play a major role to ‘guide’ the visual cortex to ‘read’ and interpret the visual information arriving from a retinal prosthesis in a blind subject. In SSD, visual information captured by an artificial receptor is delivered to the brain of a blind person using non-visual sensory information via a human-machine interface. In the present proposal, I will test this notion using, a visuo-auditory SSD, in which visual scenes are encoded into soundscapes. The work has fundamental applications in visual and cognitive neuroscience as well as practical significance for the quality of life of millions of blind people worldwide.