Rajeshwar Awatramani - US grants

[unreadable] DESCRIPTION (provided by applicant): Charcot Marie Tooth disease (CMT), in all its forms, is the most common inherited peripheral neuropathy often characterized by severe demyelination in the peripheral nervous system. In some cases it is caused by aberrant programs controlling Schwann Cell (SC) development, whereas in other cases, SC development is unimpaired but improper myelin maintenance results in late-onset neuropathy. Thus, to understand the pathomechanisms of this disease warrants a better grasp of the molecular programs underlying Schwann cell development as well as myelin maintenance. MicroRNAs, are a class of small, naturally occurring, regulatory RNAs that bind in a sequence specific manner to their cognate targets mRNAs, thereby reducing translation efficiency as well as steady state mRNA levels. These newly discovered molecules are postulated to target almost 30% of all mRNAs, and are thus likely to be involved in various stages of SC development as well as in myelin maintenance. Here we will first determine the global function of microRNAs in SC development and in myelin maintenance by eliminating the key microRNA processing enzyme, dicer. Next we will elucidate the specific microRNAs that may target mRNAs of key developmental regulators or key myelin structural proteins. These studies will open exciting possibilities for microRNA-mediated manipulations of SC gene expression as therapeutic avenues for CMT. The role of microRNAs in peripheral myelin formation and maintenance PUBLIC HEALTH RELEVANCE The proposed studies will detail the role of microRNAs as critical regulators of genes expressed in the Schwann cell lineage, and thus will have important implications for understanding the pathomechanisms of Charcot Marie Tooth Disease, and possible therapeutics. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Schwann cell differentiation is thus a multistage process, the progression from one stage to the next being driven by dynamic alterations in underlying genetic programs. We hypothesize that microRNAs, being important developmental regulators in other systems, will be critical for SC developmental transitions. To evaluate the function of microRNAs in Schwann cell (SC) development we have conditionally ablated the microRNA processing enzyme, dicer. SC lacking dicer are stalled during differentiation and do not produce myelin, phenocopying models of congenital hypomyelination. Here in Specific Aim1, we will perform a detailed ultrastructural and molecular characterization of these SC lacking dicer. Preliminary analyses suggest that these SC are arrested at the promyelinating-myelinating transition, maintain the expression of Sox2, and lack the SC master regulator gene Egr2 (Krox20). To prove that loss of Egr2 is the underlying reason for dysmyelination, we will determine if forced expression of Egr2 can circumvent the phenotype of SC lacking dicer. Finally, we will generate a mosaic deletion of dicer, to verify that the changes observed in the dicer cKO are cell autonomous. In Specific Aim 2, we will perform microRNA profiling in developmental and pathological scenarios. We will identify key microRNAs that are dynamically expressed and then bioinformatically determine their candidate targets. We will then verify microRNA-target interactions using luciferase reporter assays in heterologous cells, argonaute co-immunoprecipitation, and overexpression in SC-neuron coculture systems. Finally we will determine whether some key microRNAs are themselves activated by Egr2, testing the hypothesis that Egr2 represses antecedent gene programs in SC with the help of microRNAs. Together these studies will be important in understanding the role of microRNAs in development. Further, since the processes underlying SC differentiation are considered a mirror image of SC dedifferentiation, these studies will not only shed light on developmental transitions but also on converse transitions that occur in peripheral myelin disorders and nerve injury.

DESCRIPTION (provided by applicant): The drastic motor deficit in Parkinson's disease (PD) patients is largely caused by a substantial loss of midbrain dopamine neurons (mDA). Careful morphometric studies have revealed a selective susceptibility of certain mDA populations. Thus, mDA neurons found in the ventral tier of the substantia nigra pars compacta (SNpc; A9) are more vulnerable, compared to mDA located in the dorsal tier of the SNpc, or in the Ventral Tegmental Area (A10). This differential susceptibility highlights the diversity of mDA populations. We hypothesize that in the developing midbrain, there are multiple distinct mDA progenitor pools, each of which gives rise to distinct mDA subtypes. We will attempt to determine the progenitor pool for the most susceptible type of dopamine neuron in PD. Accordingly, we will lineage trace one proposed mDA progenitor pool and determine whether its descendents populate the most vulnerable regions of the dopaminergic field i.e. the ventral tier of the SNpc. Next we will develop topographic maps for this DA subtype. Together, these experiments will provide a first glimpse into how mDA diversity is generated. Elucidating the developmental basis for this diversity will be critical for understanding differential susceptibility of mDA, as well as generating accurate ES or iPSC stem cell derived models and therapies for PD.

? DESCRIPTION (provided by applicant): Lineage analysis serves to define progenitor-progeny relationships in embryonic development. These analyses inform our understanding of the types and timing of choices that progenitors make towards generating the complexity of cell fates observed in the adult organism. In the embryonic CNS, lineage analysis has provided a fundamental substrate on which scores of models are built upon, and have provided an understanding of how the embryonic neural tube progenitors, through a series of molecularly guided decisions, generate the enormous complexity of cell types observed in the adult CNS. All methods of lineage tracing rely on a single fundamental principle - that of being able to indelibly label a progenitor, or subset of progenitors. Most recently, this has been accomplished through the use of site- specific recombinases. Site-specific recombinases can be driven by gene-specific promoters in discrete regions of the developing neural tube. In these cells, the recombinase activates a reporter in a permanent manner, thereby permitting tracking of those cells, and their descendent lineages, through subsequent developmental stages. Recombinase based lineage analysis has transformed our understanding of embryonic CNS development. Recombinase based lineage analysis is subject to one critical limitation. The specificity of the method is wholly reliant on the specificity of the promoter/regulatory elements used to drive the recombinase. Towards improving the specificity of this method, we and others have developed intersectional and inducible methods to label more selective groups of progenitors. These improvements have led to even more refined fate maps in various regions of the embryonic CNS. Despite these improvements, one caveat remains - genes and their cognate regulatory elements are often expressed in progenitors, as well as in related or unrelated postmitotic neuron populations. Expression of recombinase drivers in postmitotic neurons obfuscates lineage analysis, as recombination occurs in the postmitotic cell regardless of which progenitor it was derived from. Thus, in such cases, progenitor-progeny relationships cannot be accurately ascertained. Here we propose to develop a new platform for lineage analysis, termed Progenitor anchored intersectional fate mapping (PRISM) which circumvents the aforementioned limitations. In Specific Aim 1, we will establish the validity of the PRISM approach. In Specific Aim 2, we will use this approach in proof-of- principle experiments to resolve the lineage of the key molecule Shh in the ventral forebrain wherein Shh is expressed contemporaneously in progenitor cells as well as in postmitotic neurons. The proposed strains add to the growing conditional toolbox for lineage analysis. Since most recombinase drivers are subject to limitations, this method will be broadly applicable for studying important lineage related questions.

? DESCRIPTION (provided by applicant): Dopamine (DA) deficiency, caused by DA neuron degeneration, underpins the devastating motor symptoms of Parkinson's disease (PD). DA neurons located in the ventral tier of the substantia nigra pars compacta (SNc), are particularly vulnerable, compared to those in the dorsal tier of the SNc or ventral tegmental area (VTA). Why these DA neurons display differential vulnerability remains enigmatic. Understanding the underlying mechanisms would shed light on degeneration as well as potential neuroprotective strategies to mitigate the disease. iPS-derived DA neurons are an important new method for modeling PD. Yet current protocols for generating DA neurons are not designed to generate specific DA subtypes, a critical requisite for modeling selective vulnerability. This gap exists because the molecular heterogeneity of midbrain DA neurons is not well understood. To elucidate the heterogeneity of DA neurons, we have recently used single cell molecular profiling, coupled with anatomical co-labeling studies, and revealed the existence of at least six distinct of DA neuron subtypes in mouse models. Here, we aim to use this knowledge to i. better understand DA neuron diversity in vivo ii. understand mechanisms that may influence the generation of DA neuron subtypes iii. derive and characterize two prominent DA neuronal subtypes, one located in the SNc and one in the VTA, from human iPS cells in a rational manner, and iv. use these DA neuron subtypes to examine selective vulnerability in the context of genetic PD mutations. In Aim1, we will examine how Wnt signaling may influence DA neuron subtype allocation. In Aim 2, having optimized the Wnt regimen, we will next use targeted gene manipulations to derive highly enriched cultures of two specific DA neuron subtypes, and then characterize those subtypes by physiological and transcriptomic approaches. Next, we will generate both DA neuron subtypes from iPS cells harboring a DJ-1 mutation and examine differential pathological effects on both, SNc as well as VTA DA neuron subtypes. In Aim 3, we will further characterize the phenotype of the two DA neuron subtypes in vivo. We will elucidate the projections, and complete transcriptomes of two murine DA neuron subtypes, taking advantage of genetically targeted mice. Information from this aim will further highlight the differences between these subtypes. Additionally, these results will feed back into Aims 1 and 2, to further optimize our DA neuron subtype derivation protocol. In sum, taking advantage of the combined expertise and extensive interactions of two labs, we propose a cohesive plan based on molecular logic, to derive distinct DA neuron subtypes from iPS cells and aim to improve modelling PD. These studies will open the future possibility of understanding the effects of a range of PD mutations on selective vulnerability.

The neurotransmitter dopamine (DA), produced by midbrain DA neurons, influences a spectrum of behaviors including motor, reward, motivation, and cognition. In accordance with these functions, DA dysfunction is prominently implicated in a wide gamut of disorders affecting tens of millions of people, including Parkinson's disease, schizophrenia, ADHD, addiction and depression. Understanding how DA neurons control all of these distinct behaviors is important for understanding and treating these neuropsychiatric diseases. The literature is dominated by anatomical classification of DA neurons based on location within the Ventral Tegmental Area (VTA) or Substantia Nigra pars compacta (SNc). Guided by an emerging literature on DA neuron heterogeneity, we hypothesize that there must exist several molecularly and functionally distinct DA types, perhaps intermingled, that could underpin the myriad functions of DA. As a first step in classifying DA neurons, the Awatramani lab developed an approach to profile single midbrain DA neurons, each for the expression of 96 key genes, using a microfluidic dynamic RT-qPCR array. Hierarchical clustering indicated that DA neurons exhibited roughly six distinct molecular barcodes, presumably indicative of at least six molecularly and functionally distinct DA subtypes. The Dombeck laboratory has developed a robust data set demonstrating functional heterogeneity of DA neurons in behaving mice. Previous models postulated that slow variations in tonic firing rates bias the system toward or away from movement, whereas phasic signaling was linked to unpredicted rewards. Using imaging in behaving mice, we showed heterogeneous expression of phasic locomotion and reward signaling in DA axons projecting to the striatum. In the dorsal striatum we found that most DA fibers displayed a phasic signal locked to the animal's cyclic accelerations during locomotion. In the ventral striatum, axonal signaling to unpredicted rewards was more prevalent. These results indicate that striatum DA release is not simply homogenous and movement permissive, but is richly heterogeneous with respect to reward and locomotion signaling. Based on these complementary data sets- molecular heterogeneity and functional heterogeneity, our goal is to correlate molecular identity with anatomy and function. In Specific Aim 1, we will define the diversity, transcriptomes, and projections of DA neuron subtypes, developing intersectional genetic tools to access DA neuron subtypes. In Specific Aim 2, we will establish the behavioral signaling properties of the genetically identified DA subtypes that project to the striatum. Thus, using a collaborative approach between two laboratories each with distinct expertise, we aim to characterize DA neuron subtypes based on their molecular, anatomic and functional properties. These studies will be vital for designing targeted therapies for the DA system. Moreover these studies will provide genetic platforms for manipulations of DA subtypes towards understanding their role in mammalian behavior.

Abstract The VTA is a central hub for two prevalent pathological conditions - chronic pain and opioid addiction. In both these conditions, the physiological properties of neurons in the VTA are significantly altered. Recent evidence suggests that in the case of opioid addiction, not all VTA neurons respond equally, and in the case of pain, different VTA neurons display complementary responses. A clear description of cell types in the VTA, their cognate projections and inputs, and which of these might be involved in these two conditions has not been well elucidated, and is essential to understand these pathological conditions. Here, we propose to determine the cellular, molecular and anatomical landscape of VTA cell types and how these are altered in chronic pain and addiction models. The VTA displays enormous cellular heterogeneity, being comprised of DA neurons, GABAergic neurons and Glutamatergic neurons, as well as non-neuronal cells. Further, among the DA neurons there is additional layer of heterogeneity, and at least four VTA DA subtypes have been demonstrated. This immense heterogeneity presents a problem for understanding VTA circuitry as well as its alterations in these conditions. Here, our goal is to disentangle the murine VTA into its constituent cellular components, towards understanding how these individual components are altered in pain and addiction. In Specific Aim 1, we will use single cell transcriptomics to analyze VTA cells in conditions of chronic pain, morphine administration, or both. We expect to first subdivide the VTA into its constituent cell types, and then evaluate the molecular changes within each cell type, in each condition. This aim will facilitate discovery of new molecular targets towards treatment of chronic pain. In Specific Aim 2, using newly developed intersectional genetic tools to access VTA cell types, we will determine the projections of several classes of VTA DA and non-DA neurons. Next, we will develop an intersectional rabies virus labeling approach to determine the inputs of distinct VTA cell types. This aim will provide a neuroanatomical foundation for understanding circuits involved in these pathological conditions. Finally, in Specific Aim 3, guided by data from Aim 1 and 2, we will identify and validate novel targets in specific DA cell types, with the goal of reversing the hypo-dopaminergic state that is characteristic of chronic pain. The results of these Aims will provide a molecular, cellular and anatomical framework for understanding VTA cell types, that will be relevant to all projects in this P50. Additionally, the discovery and validation of candidate receptors in distinct VTA cell types, will provide an excellent entry point towards developing alternatives to opioids in the management of chronic pain.

ABSTRACT ? TRANSGENIC AND TARGETED MUTAGENESIS LABORATORY The Transgenic and Targeted Mutagenesis Laboratory (TTML) is a Northwestern University-wide resource dedicated to the generation, import, recovery/rederivation, and cryopreservation of genetically engineered mice. TTML provides Lurie Cancer Center (LCC) members a broad range of services including generation of transgenic, gene edited, and targeted transgenic mouse models via microinjection; gene targeting/editing of embryonic stem (ES) cells; ES cell microinjection into blastocysts to create germline competent chimera; importation and recovery/rederivation of mouse lines from germplasm; and cryopreservation of mouse sperm and embryos. Since the last review, the core has introduced full-scale, start-to-finish mutagenesis capabilities. These changes were necessary to facilitate the development of newly emerging genome engineering technologies such as CRISPR. The TTML team works with LCC members to design customized genetic modification strategies and genotyping assays, and provides confirmed founders to investigators. These new capabilities, in addition to our traditional methods for creating transgenic and ESC derived mutant lines, enable investigators the opportunity to employ overarching approaches to genetic modification mutagenesis through the facility. The TTML facility is essential for the research programs of investigators of the Lurie Cancer Center who use mouse models to study mechanisms underlying malignancy and validate targets for translational projects. It provides the necessary infrastructure that allows the Center?s investigators access to transgenic technology that normally requires expensive microinjection equipment and skilled staff with expertise in microinjection, microsurgeries, embryo manipulation, animal husbandry, ES cell culture, and genome engineering. In addition, TTML staff provide in-house consultation and recurrent guidance on all transgenic-related technologies including CRISPR-mediated genome editing systems, transgenic and targeting vector design, appropriate screening strategies, DNA purification methods, breeding and analysis of founder mice, appropriate methods for importing mouse lines, and cryopreservation options. Since the inclusion of the TTML as a resource within the Lurie Cancer Center in 1995, Center investigators have consistently been the primary group of NU faculty utilizing the TTML, emphasizing its pivotal role in the overall research mission of the LCC.

Abstract: DA neuron degeneration, resulting in deficient DA signaling, underpins the debilitating motor symptoms of Parkinson?s disease (PD). Among DA neurons, those located in ventral tier of the substantia nigra pars compacta (SNc), are particularly vulnerable, compared to those in the dorsal tier of the SNc or ventral tegmental area (VTA). A mechanistic explanation of selective DA neuron vulnerability remains an important goal, that has been hampered in part by a lack of understanding of the intrinsic differences between DA neurons. We hypothesize that even within a single neuroanatomical cluster like the SNc, there exist DA subtypes with distinct developmental histories and intrinsic properties that may influence their vulnerability. Single cell expression profiling based DA neuron classification from our lab revealed the presence of a key SNc population defined by Sox6 and Aldh1a1 - this subtype was located in the ventral tier of the SNc, and was preferentially vulnerable in a toxin model of PD. Our preliminary data indicate that this population exists in human SNc, and is also selectively vulnerable in post-mortem PD samples. To interrogate the basis for selectively vulnerability in the SNc in depth, we next developed a set of intersectional genetic strategies, which strikingly defined a fault-line in the SNc defined by Sox6 expression, with Sox6+ cells being located ventrally and Sox6- cells forming the dorsal tier. Building on these studies, several key questions remain unanswered. Are dorsal and ventral SNc subtypes developmentally distinct? Do these neurons have different anatomical features and DA release characteristics? Is the size of arborizations of these neurons, a property linked to vulnerability, different? Are calcium fluxes and mitochondrial bioenergetic properties distinct? To answer these questions, in Aim 1, we will determine the origin of SNc neuron subtypes. We will use intersectional, subtractive, and inducible genetic approaches, as well as a novel progenitor anchored fate mapping approach (PRISM) to test the hypothesis that the dorsal and ventral tier DA neurons are developmentally distinguished by Sox6. We will then test the potential of Sox6+ medial vs Sox6- lateral floor plate progenitors to give rise to SNc neurons when transplanted into a PD model. In Aim 2, we will examine the arborization of genetically defined SNc neurons, since this feature has been linked to their vulnerability. Using a new sparse labeling tool, we will plot the projections of genetically defined SNc DA neuron subtypes, and determine the size of their arbors, and explore the molecular determinants of extensive arborization. We will also study DA release from axons and dendrites to determine if dorsal and ventral tier neurons are distinguishable by these criteria, also linked to vulnerability. In Aim 3, we will test the hypothesis that the SNc DA neuron subtypes have distinctive physiological properties and that these differences translate into differences in mitochondrial oxidant stress in somatodendritic and axonal compartments. Overall, our studies redefining the SNc based on lineage will provide important insights into selective vulnerability.