Lindsay A. Schwarz - US grants

DESCRIPTION (provided by applicant): For proper function of the mature nervous system, neurons send processes out during development that must choose their correct synaptic partners from an enormous population of cells. The importance of this process in the mammalian brain is underscored by the growing knowledge that many neurodevelopmental disorders, such as autism and epilepsy, as well as adult disorders like bipolar disorder and schizophrenia, may result from improper formation of neuronal circuits. Therefore, a full understanding of the molecules mediating synaptic partner choice is crucial for make advancements in our comprehension and treatment of these diseases. While many guidance cues have been identified that assist outgrowing axons to reach an area of the brain, it's less clear how an axon chooses its exact synaptic partner. The clustered protocadherin family of genes (Pcdhs) show great potential to fill this role in mammalian brain development. The Pcdh gene family consists of 58 genes in three gene clusters (1, 2, and 3), each encoding for a unique adhesive transmembrane protein. It has been shown that individual neurons express a wide diversity of Pcdh isoforms, and studies have suggested their importance for synapse formation and axon targeting in certain classes of neurons. Furthermore, linkage analyses of schizophrenia and bipolar disorder patients have uncovered susceptibility loci for both disorders in the human genome near the clustered protocadherin gene family. Yet progress in understanding the function of Pcdhs has been limited by lethality in Pcdh-3 knockout animals, while studies of the other clusters are few (for Pcdh-1) or non-existent (for Pcdh-2). However, by using Mosaic Analysis with Double Markers (MADM), it should be possible to gain novel insight into the mechanisms by which Pcdhs contribute to neural development. Briefly, MADM allows for simultaneous labeling and gene knockout in individual cells. By inserting the appropriate labeling cassettes into chromosome 18 and breeding these mice (called MADM18 mice) with various Pcdh heterozygous mutant mice (1, 2, and 3 mutants, or a mutant mouse where all three clusters have been deleted), it will be possible to analyze axonal projections, dendritic arborizations, and synapse formation of Pcdh-lacking neurons next to wildtype-labeled neurons in a variety of brain regions where these proteins are known to be expressed. Compared to previous Pcdh-knockout studies, this strategy has several benefits: it avoids the lethality associated with complete loss of Pcdh-3, and should provide a more accurate assessment of Pcdh function through single cell knockout of the genes. Characterizing the role of the clustered protocadherins in neuronal development may also have value for understanding the molecular mechanisms of several neurological diseases, such as autism and schizophrenia, where it's thought that improper synapse formation may be involved. Finally, the MADM mouse produced for this study could be used in the future to analyze any gene distal to the MADM cassettes on the chromosome 18, which may be a useful tool for researchers in a variety of scientific fields. PUBLIC HEALTH RELEVANCE: Initial research suggests that the clustered protocadherin gene family is crucial for nervous system development, though due to lethality in knockout mice, it has been challenging to determine their exact function. Using a novel technique (Mosaic Analysis with Double Markers or MADM), it will be possible to circumvent previous limitations and finally ascertain the specific role of Pcdhs in neurodevelopment, information that could also provide insight for neuropsychiatric disorders such as autism, schizophrenia, and bipolar disorder. Finally, the MADM mouse generated for this study can be used to analyze other genes on mouse chromosome 18, many of which are implicated in important diseases such as cancer, depression, and heart disease.

Project Summary Next-generation sequencing has enabled a rapid expansion in cellular taxonomy across biological systems. However, to probe the function of these newly defined cell types, researchers require a molecular toolkit that can precisely target cells based on a combination of features. To address this need, this project proposes the development of novel intersectional approaches that will restrict transgene expression to defined cell populations based on multiple characteristics, such as combinatorial gene expression and activity state during defined behaviors. While a handful of intersectional strategies currently exist, their limitations, such as complicated design parameters and limited spatial or temporal resolution, preclude their widespread use. The intersectional strategies proposed here address these limitations to provide two new types of tools: 1) a Cre and Flp-recombinase dependent AAV vector that is easily modified to accommodate a variety of transgenes and promoters and 2) a transgenic mouse line that restricts Cre or Flp recombinase expression to neurons activated in a tightly-defined time window (~30 minutes). Using these tools, our goal is to address a major unanswered question in neuroscience: how do discrete populations of neurons in the brain generate diverse behaviors? Towards this, we focus on a small group of neurons in the brainstem nucleus locus coeruleus (LC) that are known to regulate different forms of arousal. Despite their small number, LC neurons are the brain?s main source of norepinephrine, a neurotransmitter that promotes a wide range of behaviors related to arousal. Traditionally, the LC has been classified as molecularly homogeneous, since all neurons within it express NE. Thus, it is not clear how the LC achieves its functional diversity. We propose that distinct subsets of LC neurons, which may differ in their connectivity and molecular identity beyond NE expression, are responsible for promoting different arousal responses. Using the intersectional tools described here, combined with single cell RNA sequencing and a novel behavioral paradigm, we will identify the molecular landscape and brain-wide connectivity of discrete LC neuron populations that promote opposing types of arousal (positive or aversive). Identifying functional diversity in the LC also has implications for human health, as perturbations in LC- related neural circuits are thought to significantly contribute to mood disorders such as depression and anxiety. Understanding how LC neurons accurately generate different forms of arousal may also illuminate how particular alterations in these neural circuits promote specific mood disorder phenotypes.