John Ngai - US grants

DESCRIPTION (provided by applicant): Vertebrates recognize and discriminate thousands of odorants of diverse molecular structure. How is this process of molecular recognition accomplished? The identification of an odorant's chemical structure is thought to occur through the combinatorial integration from multiple odorant receptors, each tuned to recognize different molecular features. Thus, by elucidating the molecular specificities of the odorant receptors we will gain a better understanding how information is processed in the olfactory system. In addition, knowledge of the molecular principles underlying odorant recognition will illuminate how these receptors are tuned to bind and discriminate odorous ligands. In this application, we focus on the properties of the "C family" G protein-coupled receptors expressed in the vertebrate olfactory system. This receptor family includes the putative pheromone-sensing receptors of the mammalian vomeronasal system and the amino acid sensing receptors of the fish olfactory system. Our studies comprise three major lines of investigation using the fish as a model system: (1) We will apply and refine computational modeling approaches to elucidate the ligand-receptor interactions in two olfactory amino acid receptors expressed in the fish olfactory system. A novel "virtual high-throughput" computational screening protocol will also be implemented to identify high- affinity ligands for these receptors. Such ligands will provide useful tools for probing the molecular architecture of the receptors'ligand binding pockets. These studies will advance our understanding of the molecular determinants underlying ligand recognition in this class of chemosensory receptor. (2) Using recently developed heterologous cell-based and in vivo assay systems, we will characterize the ligand tuning properties of other members of the olfactory C family receptor repertoire. Elucidation of the receptive field properties of individual receptors in turn will allow an understanding of how multiple receptors are used in combination to recognize and discriminate odorants. (3) We will study the role of receptor multimerization in olfactory receptor trafficking and function. We will test the hypothesis that heteromeric receptor interactions are required for the localization of functional receptors to the sensory neuron's apical dendrite - the site of interaction with odorants in the external environment. These studies will establish what role, if any, receptor- receptor interactions play in the formation of functional receptors in the olfactory sensory neuron. Together our studies will help to elucidate the molecular and cellular mechanisms of olfactory coding. PUBLIC HEALTH RELEVANCE The olfactory system receives and interprets chemical cues from the environment that regulate feeding, reproduction, and other social behaviors. These chemical cues are detected by receptors expressed in the primary sensory neurons of the nose. The studies proposed in the present application will investigate the principles underlying chemical recognition by olfactory receptors, and will illuminate how these receptors regulate important physiologic and behavioral processes in humans and non-human animals.

The human brain comprises approximately 100 billion neurons precisely organized into networks that give rise to cognitive phenomena such as emotion, learning and memory, and perception. Unfortunately, there are virtually no unique markers for the many neuronal cell types thought to exist in the CNS. Indeed, we also do not know how many different cell types actually exist. Our long-term goal is to quantitate the diversity of brain regions and neuronal cell types by elucidating region- or cell type- specific patterns of gene expression - the molecular anatomy of the brain. In order to achieve this goal, we aim (1) to develop methods for mapping gene expression within identified brain regions, and (2) to develop methods for defining neuronal identity and marker genes in the vertebrate CNS. The first aim includes improving a novel mRNA amplification procedure we have developed that enables one to construct cDNA libraries from small amounts of tissue, such that the representation of original mRNA lengths is comparable to the best cDNA libraries made from unamplified RNA. Methods will also be developed for mapping gene expression within small regions of the CNS using this improved mRNA amplification procedure in DNA microarray hybridizations. The second aim includes developing methods for defining neuronal cell types and cell-type specific marker genes in heterogeneous neuronal populations. This will be done by using microarrays to characterize gene expression patterns of single cells. Methods will be developed to amplify the mRNA content of single cells to hybridize to microarrays, and statistical methods will be developed for analyzing and comparing single-cell microarray hybridization data from multiple experiments. These latter methods will allow the definition of neuronal cell identities as well as novel marker genes based upon complex gene expression patterns. Our methods to map gene expression in the CNS will enable researchers to identify genes and gene variants that are playing a role in the function of small brain regions in both healthy and diseased tissue. In addition, our methods to identify marker genes will eventually allow individual neuronal types to be isolated or targeted for study.

[unreadable] DESCRIPTION (provided by applicant): This proposal requests support for 15 trainees to continue a multidisciplinary Neuroscience Training Program for predoctoral training in neuroscience at the University of California, Berkeley. Administered under the auspices of the Helen Wills Neuroscience Institute, the goal of this Ph.D. Training Program is to educate and mentor graduate students in neuroscience, and to train them to become tomorrow's leaders as research scientists and teachers. The 35 participating faculty are drawn from across the entire campus and represent neuroscience, research from molecules and genes, to cells and circuits, systems and computation, and behavior and cognition. Training faculty include members of the Departments of Molecular and Cell Biology, Psychology, Vision Science, Chemical Engineering, Environmental Science, Policy, and Management, Integrative Biology, and Public Health. [unreadable] [unreadable] The Helen Wills Neuroscience Institute was created with the vision of training neuroscience graduate students in an interactive environment that builds bridges across traditional academic boundaries to span from genes and molecules to brain and behavior. Since its founding 5 years ago, the institute has supported the integration and expansion of neuroscience at DC Berkeley through the recruitment of new neuroscience faculty; the establishment of three technology centers - the Brain Imaging Center, Molecular Imaging Center, and Neurogenomics Center; and the establishment and growth of the Neuroscience Graduate Program - an interdepartmental Ph.D.7granting program. Together the Helen Wills Neuroscience Institute and Neuroscience Graduate Program provide the intellectual center for neuroscience research and education on the DC Berkeley campus. Students are admitted to this NIGMS Training Program mainly through the Neuroscience Graduate Program, as well as through the graduate programs of participating faculty departments. The Training Program emphasizes interdisciplinary approaches to fuel paradigm shifts in how we study the brain, with the ultimate goal of translating basic discoveries into solutions for human neurological diseases. The program provides training across the entire range of neuroscience through required course work and lab rotations that emphasize critical thinking in defined areas as well as breadth in knowledge. Students are also exposed to many facets of neuroscience research through seminar series and lectureships, a journal club, and an annual campus-wide retreat. Students supported by this NIGM Training Program have outstanding academic credentials, conduct innovative research, and publish widely in the top journals of our field. The vast majority of past trainees supported by this Training Program have gone on to productive careers in academic biomedical research. [unreadable] [unreadable] [unreadable]

PROJECT SUMMARY The generation of neuronal diversity in the nervous system requires the specification and differentiation of a multitude of cellular lineages. Successive developmental programs control the generation of individual neuronal types, cell migration, axon extension, and ultimately the formation of functional synaptic connections. The specific genetic programs underlying the differentiation of mature neurons from their progenitors remain incompletely characterized, in part because of the difficulty in studying neuronal progenitor cells in their native environments. Similarly, mechanisms enabling tissue regeneration following injury in the adult nervous sysem are incompletely understood. In the vertebrate olfactory system, primary sensory neurons and other cell types are continuously regenerated throughout adult life via the proliferation and differentiation of multipotent neural progenitor cells. This feature makes the olfactory system particularly amenable for studies on the properties of adult neural stem cells. Following injury that results in destruction of all mature neurons and support cells in the olfactory epithelium, adult stem cells are activated to reconstitute all cell types in this structure. Of particular interest are the mechanisms that support regeneration of the olfactory epithelium following injury and whether and how they differ from mechanisms subserving tissue homeostasis under normal conditions. Elucidation of these regulatory networks is critical for understanding how mature neuronal and non-neuronal cell types are generated from the adult tissue stem cell of the olfactory epithelium. In this application we propose to investigate the cellular mechanisms and genetic pathways subserving the reconstitution of the olfactory epithelium following injury. Specifically, we propose a unique suite of approaches including single cell transcriptome profiling combined with rigorous statistical analysis, in vivo lineage tracing, and genetic pertubations to (1) elucidate the mechanisms underlying injury-induced regeneration in the olfactory epithelium stem cell niche and (2) establish the roles of canonical Wnt signaling and Sox2 in early olfactory neurogenesis. Together these investigations will provide a model for understanding the mechanisms regulating other neural stem cell types and lay the groundwork for the future development of treatments and therapeutics to ameliorate neural tissue damage and degeneration.

DESCRIPTION (provided by applicant): The 454/Roche GS-FLX pyrosequencer will be used for high-throughput DNA sequencing in applications demanding long reads (>400 nt) at high accuracy. The technology and characteristics of this sequencing technology is markedly different than for sequencers such as the Solexa/Illumina Genome Sequencer, which provide short (<75 nt) read lengths with a higher error rate. The ability to obtain long read lengths with high accuracy affords several critical advantages for a number of applications, including (1) the sequencing of novel genomes, (2) EST and transcriptome characterization in organisms lacking a sequenced or well-mapped genome, and (3) the analysis of alternative pre-mRNA splicing. The 454/Roche long-read sequencing technology will be applied to a diverse array of NIH-funded projects as well as other projects of high relevance to biomedical science, including the characterization of multigene families involved in sensory perception;the elucidation of the patterns and mechanisms of alternative pre-mRNA splicing;the characterization of genomes of genetic and evolutionary models of human development and disease;an analysis of the genetic loci associated with cancer;and an investigation of the susceptibility of mammalian cells to nutritional ions and environmental toxins. There are currently no 454 DNA sequencers on the UC Berkeley campus;acquisition of this instrumentation by the FGL will support researchers pursuing these projects, which promise to illuminate the fundamental mechanisms underlying human biology and disease.

? DESCRIPTION (provided by applicant):Unraveling the complexity of the mammalian brain is one of the most challenging problems in biology today. A major goal of neuroscience is to understand how circuits of neurons and non-neuronal cells process sensory information, generate movement, and subserve memory, emotion and cognition. Elucidating the properties of neural circuits requires an understanding of the cell types that comprise these circuits and their roles in processing and integrating information. However, since the description of diverse neuronal cell types over a century ago by Ramon y Cajal, we have barely scratched the surface of understanding the diversity of cell types in the brain and how each individual cell type contributes to nervous system function. Current approaches for classifying neurons rely upon features including the differential expression of small numbers of genes, cell morphology, anatomical location, physiology, and connectivity - important descriptive properties that nonetheless are insufficient to fully describe or predict the vast number of different cell types that comprise the mammalian brain. Here we propose a suite of technologies for identifying and classifying the myriad cell types present in the brain. Our method will be developed using layer 5 pyramidal cells from mouse somatosensory cortex as a model system. First, we will exploit the latest developments in DNA sequencing technologies to characterize gene expression profiles on single layer 5 neurons at high throughput. This information will be used to classify individual cells based on their transcriptome fingerprints. Second, genes found to define newly discovered neuronal subtypes will be used to gain genetic access to these cells using Cas9/CRISPR-mediated genome engineering to create transgenic reporter lines. Development of this technology promises to open a pipeline for the rapid generation of multigenic mouse reporter strains in which specific neuronal subtypes are uniquely labeled by combinations of tagged genes. Third, we will use these genetically engineered mice to confirm that our taxonomy represents distinct functional properties of the classified neurons. Our approach can ultimately be scaled up to generate a complete census of cell types in the brain, a critically needed resource for dissecting nervous system function with modern investigative tools.

PROJECT SUMMARY Understanding how the nervous system extracts and encodes features of an organism?s environment is a major goal of sensory biology and systems neuroscience. In the vertebrate olfactory system, projection neurons of the main olfactory bulb receive synaptic input from the primary sensory neurons in the peripheral olfactory epithelium. These projection neurons ? comprising the mitral and tufted cells ? relay sensory information to multiple higher order brain centers to drive a spectrum of innate and learned behaviors. Previous studies indicate that olfactory bulb projection neurons are functionally if not genetically heterogeneous: the mitral and tufted cells are anatomically, morphologically and physiologically distinct; mitral cells can be distinguished based on their differential expression of ion channel subunits and intrinsic physiological properties; and projection neurons display different patterns of innervation in higher order olfactory centers implicated in innate and learned olfactory behaviors. One view based on this large body of evidence is that subtypes of projection neurons represent different features of the odorant stimulus and carry parallel streams of information to different olfactory centers to generate behaviors. While electrophysiological properties, expression of selected molecular markers and connectivity have been used to describe differences among projection neurons, current approaches have not yet yielded a systematic classification of olfactory bulb projection neuron diversity. This information is necessary for elucidating in a comprehensive manner the principles underlying olfactory coding as information is relayed and transformed by the population of olfactory bulb projection neurons. In the present application, we propose to apply single cell RNA profiling by deep sequencing (single cell RNA-Seq) to generate a taxonomy of olfactory bulb projection neurons. The single cell approach relies upon each cell type?s unique transcriptional signature to define itself and therefore requires no a priori knowledge of the nature or extent of diversity within the population. The taxonomy of projection neurons will be independently validated by RNA in situ hybridizations. We will further test the hypothesis that projection neurons innervating different brain regions are genetically distinct. Importantly, the identification of cell type-specific genes will allow genetic access to the newly identified cell types and the circuits in which they function. Thus we expect the proposed R21 project to enable the future development of powerful genetic tools for targeting the subcircuits mediating specific olfactory behaviors.

PROJECT ABSTRACT Major challenges in the study and treatment of Alzheimer?s Disease (AD) include the pressing need to illuminate the causative molecular mechanisms underlying this disease and to identify robust biomarkers that can predict an individual?s likelihood of developing AD prior to the onset of symptoms. Previous studies have shown an age-related decline in olfactory sensory function in humans. Intriguingly, olfactory dysfunction is also associated with degenerative conditions including AD and in some cases can predict in healthy individuals whether they will later develop AD. The molecular, cellular and anatomic underpinnings of these observations have yet to be fully characterized, however. Working under the scope of the parent grant, in this supplement we plan to use single-cell RNA-sequencing to extend our characterization of olfactory horizontal basal cells (HBCs) ? the deep reserve stem cells of the olfactory epithelium ? and their regenerative capacity to include an analysis during normal aging. We will also use single-cell RNA-sequencing to search for molecular changes associated with early stages of degeneration in a mouse model of AD. Our overall goal is to identify the molecular basis for age-associated decline in olfactory function and understand the association between anosmia and AD. The research proposed in this supplemental application also has the potential to identify early stage molecular biomarkers for AD. Further, by incorporating an analysis of the molecular changes associated with age-related decline in olfactory function we expect to reveal the mechanisms and pathways driving early disease progression itself in AD and other neurodegenerative diseases.