During neuronal differentiation, precursor cells must acquire their definitive functional transcriptomes. Regulation of transcriptional expression of several genes as well as of post-transcriptional processing, especially alternative splicing (AS), is crucial to this cellular process. Importantly, these layers of transcriptomic regulation are interconnected. On the one hand, changes in the transcription of neuron-specific splicing factors have great effect in modulating many alternative exons. On the other hand, several studies point to a regulation of transcription factor (TF) function by AS, which may have a profound impact in the regulation of the associated TF target networks. However, despite some examples, the extent and architecture of this crosstalk between layers largely remain unknown. The goal of this proposal is thus to assess the impact of AS of TFs in the process of neuronal differentiation, by pursuing the following specific aims: (1) Build a comprehensive database of alternatively spliced exons in TFs; (2) Study the regulation of TF’s alternative exons during neuronal differentiation, using RNA-seq; (3) Investigate the specific functions of 10-15 regulated alternative exons, using isoform-specific knockdowns in several neuronal differentiation assays; and (4) Elucidate isoform-specific networks of regulated targets, using isoform-specific ChIP-seq and microarray quantifications. We will focus on cases in which AS is predicted to affect TF DNA-binding specificity/affinity or cofactor interactions. These are expected to have a profound impact in which targets each isoform regulates, challenging the way we think of the regulation exerted by a single TF gene.

Bone marrow is an organ where various kinds of hematopoietic cells reside and undergo maturation or differentiation into specific lineages, processes that occur in specialized locations called 'niches'. Whereas other hematopoietic and lymphoid organs, such as lymph nodes and spleen, have definite inner substructures that are easily identifiable because of the distinct histological architecture, such substructures are not as obvious in the bone marrow space. However, the requirements regarding process specificity in bone marrow are at least as high as in the other hematopoietic and lymphoid organs. Even though there is ample indirect evidence of the existence of such specific substructures that operate as functional niches, it is still not possible to clearly distinguish which areas of the bone marrow correspond to, for instance, the "B cell niche", the "osteoclast niche" or the "hematopoietic stem cell (HSC) niche". Previous studies have been interpreted as indicating that the HSC niche is located along endosteum close to osteoblasts, or around the vasculature. However, these concepts derive only from static analyses using conventional immunohistochemistry and/or adoptive transfer experiments and thus do not allow for a comprehensive investigation of the complex dynamic biological processes required for the functional characterization and understanding of the different niches. The purpose of this study therefore is to visualize and identify the distinct niches in bone marrow cavities by performing cross-disciplinary studies that combine advanced imaging technology with novel computational techniques. Imaging mass spectrometry will be exploited to identify molecules specific for certain niches, whereas the dynamics of bone marrow cell behavior in the respective niches will be analyzed in detail by using intravital two-photon bone imaging that the applicant has originally pioneered. By combining these advanced imaging approaches with novel high-throughput computational image analysis of cellular migration and interaction dynamics within the native tissue context, and then integrating all the results in multicellular simulation models, we believe our team will be able to anatomically and functionally define the different "niche" substructures in bone marrow cavities in vivo with greater resolution and assurance than previously possible.

Cortical interneurons are remarkably diverse in their properties. Despite a growing appreciation of the role played by sonic hedgehog (Shh) and fibroblast growth factor (FGF) signaling in patterning the ventral telencephalon, little is known about how these pathways and their downstream effectors influence the emergence of interneuron diversity later during telencephalic development. The proposal aims to further elucidate the transcriptional cascade involved in specifying different interneuron subtypes focusing on genes acting downstream of FGF. I propose to study two Ets-domain transcription factors (TF), Ets1 and Ets2, known to act downstream of FGF signaling upon activation through MAP kinases, and recently identified as highly expressed in both caudal and medial ganglionic eminences (CGE, MGE). I speculate that the Ets-TFs might contribute to interneuron subtypes specification. I intend to investigate the regulation of Ets-TFs in vivo in both GE through FGF signaling-dependant phosphorylation, as well as their role in the specification of cortical interneurons using the Ets1 and Ets2 null mice, through electrophysiology and morphological analyzes. Finally, I intend to explore the consequences of Ets1 or -2 genetic inactivation on mice behavior and locomotor activity. This transversal project will attempt to better understand the molecular basis of interneuron development and specification as well as the contribution of specific interneuron subtypes to behavior, to further increase our knowledge of forms of neurological and neuropsychiatric disorders (epilepsy, autism spectrum disorders, schizophrenia) in which specific populations of interneurons have been implicated.

Type 2 diabetes mellitus (T2DM) and obesity are leading causes of mortality and are associated with the Western lifestyle, which is characterized by excessive nutritional intake and lack of exercise. A central player in the pathophysiology of these diseases is the nuclear hormone receptor PPARgamma (PPARg), a lipid sensor and master regulator of adipogenesis. PPARg is also the molecular target for the Thiazolidinedione (TZD)-class of insulin sensitizers, which command a large share of the current oral anti-diabetic drug market. Despite their efficacy in glycemic control, TZDs are associated with various adverse side effects, including weight gain, edema, and liver and cardiovascular toxicity, leaving room for novel therapeutic approaches that target this pathway.
During the course of my HFSP long-term fellowship I have identified gamma activated peptide 1 (GAP1) as a novel PPARg target. In experiments with GAP1 knockout mice, we further demonstrated that these mice develop high-fat diet induced insulin resistance and hepatic steatosis, underscoring its significance in the development of T2DM.
Basis for the current proposal is our finding that administration of GAP1 to diabetic mice has potent and long lasting beneficial metabolic effects, including lowering of blood glucose and insulin, similar to TZDs. The studies proposed here aim to mechanistically understand the pharmacologic effects of GAP1 on glucose homeostasis and the development of T2DM. Therapeutic targeting of GAP1 could serve as an improved strategy for the treatment of T2DM, eliminating some of the adverse effects of TZDs that are mediated through direct activation of PPARg.

Circadian clocks keep temporal order and help organism to anticipate daily changes in the environment. In mammals, most, if not all, differentiated cell types possess a circadian clock, with over 10% of their transcriptome changing throughout the day. This is achieved through a network of activators and repressors that form interconnected feedback loops. Cells likely acquire circadian rhythms only upon differentiation, since the only cell systems in which circadian rhythms have not been found are embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs). Thus, pluripotent cells either cannot sustain a circadian clock, or the clock they possess neither depends nor causes transcriptional oscillations. As a result, pluripotent cells may offer an ideal system to develop a model for the emergence and maintenance of the circadian clock, and to study how key properties of these timing devices – such as robustness, plasticity, and temperature compensation -- are implemented into the system.

Here, we aim to decipher the molecular and cellular mechanisms behind the generation of the circadian clock. We will use innovative and interdisciplinary experimental and analytical approaches to dissect the activation of the transcriptional circadian cycle during ESC differentiation, as well as its deactivation during somatic cell reprogramming to iPSCs. We will build on our team’s expertise in circadian rhythms and RNA biology (SK), ESCs, differentiation and reprogramming (EM) and systems biology (AR). We will analyze circadian gene expression during the development or suppression of the circadian system using both live imaging-based single cell assays and genome-wide transcriptome sequencing (RNA-Seq). We will use computational algorithms to integrate the data into a system scale mechanistic model of the initiation and shut down of the circadian clock. We will further validate and refine the model by assessing these processes under genetic perturbation of key system components as well as by biochemical experiments (e.g. ChIP-Seq) in differentiated and pluripotent cells to test the regulatory connections in our model. Our work will provide a comprehensive view of a clock as a biological system, and will shed light on the initiation of the circadian clock during differentiation and on the role of this timing device during the exit from pluripotency. Our approach harnesses the power of two distinct facets of systems biology – single cell studies of system function and genomic studies of systems organization – to tackle a fundamental question in circadian biology, and can serve as a paradigm for similar studies of other biological systems.

Species remain unaltered over millions of years yet can rapidly adapt to changing conditions. Compelling evidence from prokaryotic biology, vertebrate immunoglobulin diversification, and tumorigenesis has demonstrated that compromising genome integrity can be of adaptive value. Although stress-inducible genome instability has been described in many organisms, the molecular mechanisms underlying these phenomena in eukaryotes remain unknown. Given the links between the heat-shock protein (Hsp) Hsp90 and genome maintenance, I hypothesize that Hsp90 can act as a “molecular rheostat” to adjust the fidelity of DNA transactions in response to environmental cues, with the net effect that when an organism is not well adapted to its environment new traits will appear at higher rates that when it is well adapted. This project seeks to elucidate the mechanistic roles of Hsp90 in genome maintenance in S.cerevisiae, by characterizing key signal transduction cascades that act in coordination with Hsp90 upon stress. For this purpose, I will first undertake a systematic survey for DNA damage sensitivities and signatures of genomic instability that are released upon Hsp90 inhibition. Secondly, I will employ quantitative high-throughput biochemical approaches to dissect the effects of DNA damage signaling on key Hsp90-client physical interactions. Finally, I will characterize the signaling cascades and posttranslational modifications involved and evaluate their impact on genome integrity. This work will provide insights into the biological mechanisms that drive evolution and tumorigenesis.

Every day we are assaulted by ionizing radiation (IR), in addition to clinical exposure, both of which induce significant pro-inflammatory cytokine responses. IR induction of pro-inflammatory responses can be quite severe and compromise radiation therapy, necessitating use of anti-inflammatory cortico-steroids, often with limited effectiveness. The AUF1 protein is a major attenuator of these responses through its targeted rapid destruction of pro-inflammatory cytokine mRNAs. Loss of AUF1 in mice promotes acute psoriasis, loss of follicular B lymphocytes, septic shock, and surprisingly, premature aging, cellular senescence and telomere loss, all also linked to effects of IR. Unpublished studies demonstrate that AUF1 is a key target inactivated by IR, providing an exciting molecular link between IR and inflammatory disorders. It is important to understand how IR promotes a pathological inflammatory response, for little is known about the mechanism by which this occurs. This application is directed at understanding the molecular and mechanistic basis by which AUF1 is targeted for inactivation by IR, and its role in pathological inflammatory responses. I will use immortalized wild type and AUF1-/- macrophages from the AUF1 knockout mouse and mass spectrometry, to identify and functionally characterize IR-inducible AUF1 interacting proteins. Studies have already found an exciting connection to the microRNA processing machinery and AUF1. I will also identify the major inflammatory cytokine mRNA targets regulated by IR through control of AUF1 activity. These studies forge an entirely novel area of research in understanding inflammatory disorders and environmental interactions.

At the molecular level no cell is like any other. Differences between genetically identical cells can in fact be quite pronounced, and these differences can have profound biological and biomedical implications. Stem cells divide and differentiate in response to physiological and environmental cues but exhibit considerable differences in how they do so; equally, tumour cells respond differently to drug treatment or radiation therapy. The mechanisms driving such variability among to all intents and purposes seemingly identical cells are only poorly understood. In order to understand these fundamental processes – which also have numerous biomedical and pharmacological implications – we have to overcome experimental and mathematical modelling challenges.

Here we develop the necessary experimental and theoretical frameworks, which allow us to probe single cells, measure the abundances of key signalling molecules, and infer mathematical mechanistic models for signal transduction. We will then analyze these mathematical models in order to understand better how the structure of the signal transduction network influences cell-to-cell variability. We will then test these models and their resulting predictions in further experiments. Most importantly, however, we will develop novel strategies which will allow us to guide cellular decision making processes and determine patterns of differences between cells. If we manage to reduce or otherwise affect such variability then we gain a novel way to guide the behaviour of cells. The scope for applying such techniques is enormous and could, for example, include novel therapeutic interventions, progress in regenerative medicine, as well as better understanding of stem cells and their behaviour.

At the same time we will also investigate what determines the behaviour of cells more generally. Often cells exhibit high levels of robustness against external perturbations. We will use the mathematical models inferred for single cells in order to generate hypotheses as to which molecules, molecular processes and interactions affect such robustness and how this can be interfered with.

Among the various lipid species, fatty acyls represents the major lipid building block of complex lipids, such as a component of membrane phospholipid and a precursor of lipid mediator. Phospholipase A hydrolyzes the fatty acyls at the sn-1 and sn-2 positions of phospholipids, while acyltransferase incorporates the fatty acyls into the lysophospholipids. The dynamic equilibrium between deacylation and reacylation by these enzymes, known as phospholipid remodeling, maintains the membrane stability and the cellular function normally. Under the pathophysiological conditions, eicosanoids, the lipid mediators derived from arachidonic acids, are produced by a sequential actions of eicosanoid synthesizing enzymes. However, it is still unclear the molecular basis of the interconnectedness between eicosanoid production and phospholipid remodeling in inflammation. I will address the question by examining the kinetics of their metabolism and exposing the relationship between signaling pathway and lipid metabolism. Focusing on inflammatory cells like macrophages and mast cells may allow us to address the question because of the existence of a series of enzymes and their important roles in inflammation. I plan to a) apply the deuterium and radioisotope-labeled lipid species to monitor the dynamics of lipids in the cells, b) measure the lipid levels by a liquid chromatography-mass spectrometry system, and c) use computational modeling to understand the underlying mechanism of lipid metabolism. This study will create a bridge between lipid metabolism and inflammation. The results may also contribute towards developing an innovative drugs against inflammation and related diseases.

A fundamental aspect of cell division is the proper segregation of chromosomes in a spatially oriented manner. While the essential mechanisms of cell division are conserved in eukaryotes, additional mechanisms may exist in human cells to achieve proper differentiation, for example in asymmetrically dividing cells.

The orientation of the cell division axis is determined by forces generated at the cell cortex, where astral microtubules (MTs) emanating from the mitotic spindle pole are anchored to the plasma membrane. The minus end directed microtubule-based motor cytoplasmic dynein is a key player in generating force at the cell cortex by pulling on astral MTs. Cortical dynein functions for spindle positioning, orientation, and anaphase spindle elongation. However, how cortical dynein is targeted and regulated to achieve these different tasks remains unclear. Cortical dynein must function during both symmetric and asymmetric cell division to generate cell type diversity. However, whether cortical dynein is differently regulated between these different types of cell division and how cortical dynein functions during differentiation in human cells are unknown.

For this proposal, I will:
1: Identify dynein interacting partners at the cell cortex
2: Define the regulation of dynein localization and function at the cell cortex
3: Analyze cortical dynein localization and binding partners during differentiation in human ES cells

This project is completely distinct from my previous work on kinetochore function and will allow me to learn new ideas, techniques, and systems, including mass spectrometry, in vitro biochemistry, and ES cells, which will drive my future research.

Sensation of mechanical forces is crucial for all living organisms from bacteria to vertebrates. In metazoans, the sensation of external forces is important for hearing, touch sensitivity and pain response. The nematode Caenorhabditis elegans is an excellent model system to study mechanotransduction during touch sensation because many genes involved therein have been identified. Despite the knowledge of the molecular players participating in the touch reception, it is not known how mechanical forces lead to mechanosensory transduction (MeT) channel activation and depolarization of the membrane. This lack of knowledge is mainly due to the absence of appropriate tools to exert well-controlled forces and to study their influence on touch receptor neurons (TRNs).
Using a combination of Micro-Electro-Mechanical-Systems and elastic substrates to exert forces onto cultured TRNs, confocal imaging and patch clamp electrophysiology to study their consequences I propose to characterize how mechanical force activates electrical signal in isolated touch receptor neurons. Different scenarios of MeT channel opening will be revisited and based on these findings a mechanical model of MeT channel gating will be proposed. These results will contribute to our understanding on cellular mechanotransduction in general and how touch is sensed in particular and might also help to understand neuropathies such as allodynia and chronic pain.

Plants regulate intake of CO2 and transpirational water loss by a highly dynamic and specific cell type, the guard cell. Since fresh water has become a globally limited resource scientific attention is focusing on understanding signal transduction mechanisms and pathways in guard cells in recent years.
To adjust the water evaporation through the stomatal pore, the surrounding guard cells regulate their turgor. This is achieved by the production or degradation of the main anionic osmolyte, malate. While an astonishing scientific progress has been made in understanding abscisic acid and Ca2+ driven signal transduction, in the identification of ion channels and initial malate transporters, very little is known about the genetics of malate metabolism mechanism. This gap of knowledge underlines the need for direct genetic identification and characterizations, to understand the molecular processes of malate metabolism and regulation in guard cells.
In my project I propose to identify the source of malate and its metabolic rate limiting enzymatic steps. Beyond that I will discover the mechanisms by which the physiological stimuli abscisic acid and CO2 control malate metabolism in guard cells. Newly developed tools of Prof. Julian Schroeder and his group open now the possibility to perform guard cell specific gene silencing or expression. Together with the highly-developed tools for guard cell signaling available in the Schroeder Lab I will identify the mechanisms mediating the essential control of malate metabolism during stomatal movements. These research discoveries may also lead to new avenues for future enhancement of water use efficiency and drought tolerance of plants.

Ribbon synapses are specialized for transmission of signals in sensory systems such as the retina. They are distinguished by ribbon-like structures at the active zone. The function, assembly and targeting of ribbons to active zones are not yet well understood. As such, our broad aim is to investigate the structure, assembly and function of ribbon synapses in the mature and developing mouse retina. We will focus on ribbon synapses of photoreceptors and bipolar cells which collectively are essential for relaying light information from the retina to the brain. We propose a highly interdisciplinary approach, combining the technical expertise of the investigators which include optical imaging, electrophysiology and molecular analysis. We will specifically address three key areas:

The first area concerns the assembly and maintenance of ribbons at synaptic sites. To date, is not known how RIBEYE, an essential component of ribbons, is dynamically transported and assembled into ribbon-like structures at active zones during development. Moreover, the cellular and molecular mechanisms underlying the maintenance of synaptic ribbons in mature circuits have yet to be determined. Thus, monitoring RIBEYE trafficking in real-time and in situ is important not only for increasing our basic understanding of the normal assembly and maintenance of ribbons, but knowledge gained from such dynamic views will help interpret the effects of perturbing RIBEYE-associated molecular interactions at synapses. In Aim 1, we propose to investigate how ribbons are trafficked, targeted and maintained at retinal synapses by generating novel transgenic animals expressing fluorescently-tagged RIBEYE and using live-cell imaging approaches (RW,FS,LL).

Second, some retinal synapses are functional during development prior to the assembly of ribbons at the active zones. How synaptic transmission changes before and after ribbons form is unclear. Knowledge obtained from physiological experiments addressing this question will advance our understanding of the function of ribbon-mediated transmission at sensory synapses. Thus, in Aim 2, we seek to understand how properties of synaptic transmission alter as ribbon synapses form and mature at inner and outer retinal synapses. Synaptic function will be assessed at different structural stages by electrophysiology (LL,RW,FS) and by two-photon imaging of synaptically-localized reporters of neurotransmission expressed in new transgenic mouse lines (LL,RW).

Third, ribbon function in neurotransmission has thus far been largely studied in mutants with altered transmitter release due to perturbed exocytosis. The Schmitz lab has discovered a novel function for the B-domain of RIBEYE itself, potentially affecting endocytosis. In Aim 3, we will determine the role of the enzymatic activity of RIBEYE(B) in the formation, maintenance and function of ribbon synapses using a knockout mouse (FS,RW & LL).

Increasing evidences indicate prolyl isomerase Pin1 regulates key proteins in phosphorylation signaling especially related to proliferation and transformation. In many cancers, Pin1 is overexpressed and correlates with poor clinical outcome. Transgenic mice studies demonstrate Pin1 knockout effectively prevents oncogene-induced breast cancer, and MMTV-Pin1 transgenic mice develop hyperplasia of mammary epithelial cells and breast cancer. A small subset of cells referred as cancer stem cells have been implicated to be responsible for tumor initiation, distant metastasis and drug resistance. However, the role of Pin1 in neoplastic stem cell is not known. My objectives are to determine the function and regulation of Pin1 in normal and neoplastic mammary stem cells. Aim 1 is to investigate the role of Pin1 in self-renewal and lineage-specific differentiation of normal mammary stem cells in vitro and in vivo using Pin1 knockout mice and MMTV-Pin1 transgenic mice that we generated. Aim 2 is to investigate role of Pin1 in breast cancer stem cells in vitro using breast cancer cell models, and in vivo by monitoring breast cancer stem cell population and the breast cancer development using Neu mouse model with Pin1 knockout or overexpression. Aim 3 is to determine the role of miRNAs in regulating Pin1 function in stem cells. This is especially exciting given miRNA-200c downregulation links breast cancer stem cell with normal stem cell and that I found several microRNAs including miR-200c target Pin1 mRNA. Results from proposed study would provide significant novel insight into the turmorigenesis of breast cancer and might help design more effective therapies against breast cancer.

KATP channels are hetero-octameric potassium selective ion channels that produce ATP-sensitive currents. Their activity is inhibited or potentiated via direct binding of intracellular ATP and ADP, respectively. Thus by functioning as barometers of cellular energetic status, KATP channels bridge metabolism to electrical activity. This property allows KATP channels to coordinate physiological processes ranging from hormone secretion to the regulation of cardiac function. Mutations associated with important human diseases such as diabetes have been mapped to genes encoding protein subunits that constitute KATP channels. Coincidently, KATP channels are targeted by anti-diabetic and anti-hypertensive drugs. As such, there is a pressing need to better understand KATP channel function, yet many details of how they operate remain obscure.

The research project I propose seeks to uncover the basis of KATP channel regulation by nucleotides. To understand how ATP inhibits KATP channels, I will solve crystal structures of the KATP channel with and without ATP. To understand how ADP potentiates KATP channel function, I will solve crystal structures of KATP channels with or without ADP. I will use electrophysiological and biophysical approaches to further probe how nucleotide binding modulates KATP channel function. These studies should reveal the basis of nucleotide recognition and how nucleotide engagement controls KATP channel activity.

Insights gained from this project may explain how disease-causing mutations in KATP channel proteins cripple its function. This project also sets the stage to investigate the mode of action of anti-diabetic and anti-hypertensive drugs.

Epigenetics refers to covalent modifications of the DNA and its associated proteins. By regulating chromatin structure, epigenetic modifications play an essential role in controlling access to genes and regulatory elements in the genome. In addition to their critical roles in development and lineage-specification, epigenetic mechanisms contribute significantly to disease pathology, including genome-wide loss and aberrant local gain of DNA methylation in cancer. Epigenetic deregulation has also been implicated in congenital disorders, and may also provide a link between early environmental exposures and susceptibility to metabolic diseases such as diabetes later in life. The frequency of hematopoietic diseases increases dramatically with age and we hypothesize that alterations in DNA methylation play an important mechanistic role.

Our goal is to determine the cause, extent and consequence of DNA methylation changes during aging. As a disease relevant and well-defined lineage we will initially characterize genome-wide DNA methylation patterns in highly purified HSCs, progenitor and differentiated cell populations at different ages. We will apply in vitro culture models to determine the mechanism and key components that might lead to alterations in vivo. This work should eventually lead to a better understanding of the cause of age-depended epigenetic alterations and more targeted treatment strategies.

Haematopoietic stem cells (HSC) are the source for life-long regeneration of immune and blood cells thanks to their ability to balance proliferation and quiescence, differentiation and self-renewal. Much evidence has been accumulated about the role of extrinsic signals in regulating HSC and the scientific concept of the stem cell niche refers to the physical and functional environment responsible for the integration of such signals. Despite the long tradition of haematopoietic research, the organizational principles underlying the spatial arrangement of the niche components, e.g. cells, extracellular matrix, soluble factors, and their impact on HSC localization, function, and fate are still incompletely understood.

Due to its complexity, HSC–niche interactions cannot be fully tackled by a single methodology. An interdisciplinary, integrated approach is needed to obtain a systemic characterization of the spatio-temporal control of HSC localization within the bone marrow (BM) and its impact on stem cell fate. We propose an iterative strategy, implementing in vivo and in vitro data from specifically designed experiments into the development of mathematical models that investigate regulatory principles in silico. In particular, we will use 3D in vivo imaging and cell tracking in the murine calvarium to provide a realistic picture of the spatial arrangement of HSC and BM components. Furthermore, specific regulator molecules will be identified by a novel high-throughput PCR technology allowing for screening of HSC responses to cell–cell/cell–molecule interactions. Long-term single HSC tracking in bioengineered in vitro environments will add high-content data on HSC localization, migration, and function in response to different regulator gradients. Together, these data will provide the basis for the development of mathematical models of defined HSC–niche interactions that allow to quantitatively predict the dynamical properties of these interactions and the effect of spatial niche organization.

Our interdisciplinary approach will lead to the first quantitative model of the 4D dynamics (i.e., organization in space and time) of cellular and biochemical components determining HSC fate in the BM, opening the door to predictive targeting on the HSC niche for novel therapeutic applications.

Although the acquisition, consolidation, and reconsolidation of information is the subject of a considerable amount of study in neuroscience, the mechanisms that determine how this information is allocated to specific cells in networks in the brain are poorly understood. Previous results have shown that only a portion of neurons potentially involved in encoding a memory subsequently become part of the memory trace (Repa J.C. et al. 2001; Rumpel S. et al. 2005). The study of how memories are allocated to subsets of neurons has only recently begun (Han J.H. et al. 2007; Han J.H. et al. 2009; Silva A.J. et al. 2009; Zhou Y. et al. 2009). The results suggest that the transcription factor CREB plays an important role in determining which neurons encode a given memory in the lateral amygdala (Han J.H. et al. 2007). CREB has been shown to have a critical role in long-term memory in both vertebrates and invertebrates (Silva A.J. et al. 1998). Here, I propose to test whether CREB has a role in remote memory allocation for contextual conditioning in V2, a region of the visual cortex previously implicated in remote memory. The use of lentivirus will allow me to modulate the levels of CREB in a subset of neurons and a cell-specific inactivation approach (allatostatin system), as well as the immediate early gene ARC, will allow me to determine whether these CREB over expressing cells are preferentially recruited into a remote memory trace for contextual conditioning. Finally, I plan to utilize electrophysiological recordings of specific cells in V2 involved in remote memory to explore the mechanisms of this form of memory.

Serotonin is a major neuromodulator implicated in a wide range of brain functions. Consequently, numerous hypotheses have been put forward to explain serotonergic system functioning, and the emergence in the near future of a single "unifying theory" seems unlikely. Nevertheless, a recurrent notion seems to emerge out of many studies, namely, that serotonin modulates sensorimotor processing, and this will be the focus of the proposed research. As a model system for this study, the rodent olfactory system appears eminently suitable for three major reasons: (1) this system, which is arguably the most behaviorally relevant modality of rodents, is a mediator of complex cognitive functions; (2) the olfactory bulb, in which we intend to study serotonin's effects, receives massive serotonergic input; (3) sniffing, the increased respiratory activity associated with odor sampling, is known to be modulated by serotonin. In view of these well-established facts, the serotonergic system seems to be ideally positioned to coordinate both sensory and motor aspects of olfaction. To address these issues we propose an integrative, multidisciplinary strategy, combining pharmacological, electrophysiological, behavioral, and genetic approaches. Importantly, we intend to use the newly-developed optogenetic technique to achieve the required levels of cell-type and temporal resolutions. By controlling serotonin release in the olfactory bulb of rats as they discriminate between various odors, we expect to be able to establish causal links between serotonergic activity on the one hand and perception and behavior on the other.

The endoplasmic reticulum (ER) serves many general functions, including the facilitation of protein folding and the transport of synthesized proteins, but it also has an important role in sensing cellular stress. ER-stress identifies a group of signals that induce a transcriptional program, known as the ER-stress response, enabling cells to survive protein overload and injury in the ER. This highly coordinated response involves three parallel signaling branches localized at the ER, namely IRE1, ATF6 and PERK.
The mechanisms involved in the activation of these ER-signaling pathways are poorly understood. We recently found that deficiency in a member of a newly identified family of aspartic proteases potentiates the selective activation of ER-signaling pathways in absence of an ER-stress response.
The long-term goals of this proposal are: to identify the function of this evolutionary conserved aspartic protease and determine its role in regulating ER homeostasis. Two complementary research sub-projects were indeed designed to provide a comprehensive study of molecular mechanisms and to address the physiological role of this aspartic protease in regulating ER-signaling pathways. The first sub-project will characterize the function of this protease, by focusing on the biochemical characterization of its proteolytic activity. The second sub-project is aimed at identifying its function in vivo.
The knowledge gained from this study will provide a better understanding of fundamental pathways controlling ER-homeostasis and will contribute to highlight new ER-signaling pathways functions independent of the ER-stress response.