Daniel N. Cox, Ph.D. - US grants

DESCRIPTION (provided by applicant): Elucidating the cellular and molecular mechanisms underlying class-specific dendritogenesis is important morphologically, as proper dendrite development is essential for the establishment and maintenance of functional neural circuitry, as well as in relation to neurological and neurodegenerative disease in which dendritic abnormalities may manifest as impaired cognitive function. Drosophila melanogaster has emerged as a powerful model for dissecting these mechanisms. While significant evidence demonstrates that complex transcriptional regulatory programs function to generate cell-type specific dendritic morphologies, what remains poorly understood is the downstream implementation of these programs and what cellular, molecular and biological processes are recruited to enable these changes in dendrite morphology. The current proposal will address this knowledge gap by focusing on key downstream effectors by which the evolutionarily conserved Cut homeodomain transcription factor mediates class-specific dendrite arborization and homeostasis. We will test three central hypotheses: 1) that Cut differentially regulates class specific sensory neuron dendrite arborization and homeostasis via regulation of the basal autophagy pathway; 2) that a novel zinc-finger BED- type protein encoded by CG3995 functions as a downstream efector of Cut and functionally interacts with ribosomal proteins to direct cell-type specific dendrite morphogenesis; and 3) that the evolutionarily conserved Hox proteins, Antennapedia and Sex Combs Reduced, function with Cut to differentially mediate cell-type specific dendritic morphologies. The short-term impact of the proposed studies will be novel insight into the cellular and molecular machinery by which transcriptional control exerts effects on differential dendrite morphogenesis in Drosophila. Ultimately, these studies have the potential to identify evolutionarily conserved regulatory mechanisms that govern dendrite development/homeostasis in humans and potentially contribute to our understanding of how derangements in these cellular processes may underlie neurological and neurodegenerative disease states. PUBLIC HEALTH RELEVANCE: Dendrite are primarily specialized to receive and process neuronal inputs and thus the molecular mechanisms that drive dendritic morphology are critical to establishing and maintaining a functional nervous system. This functional role is illustrated in a diverse array of neuropathological and neurodegenerative disease states including Alzheimer's, mental retardation, and Autism in which strong neuroanatomical correlates exist between dendrite defects and cognitive impairments. The proposed studies aim to elucidate key molecular and cellular programs that function in directing and maintaining cell-type specific dendrite morphogenesis and homeostasis.

DESCRIPTION (provided by applicant): Background: Dendritic arbor shape and functional properties emerge from the interaction of many complex developmental processes. It is now accepted that multiple local-level interactions of cytoskeleton elements direct the growth and development of the dendrite arbor. However, the specific mechanisms that control developmental acquisition of final functional dendritic properties are largely unknown. Addressing this fundamental question requires novel data driven systems-biology tools to study developmental and biophysical mechanisms in the same neuronal model. A tightly-knit collaboration between molecular genetics, quantitative morphometry, and mathematical simulation can for the first time enable large-scale studies capable of achieving holistic understanding of the mechanisms underlying emergent features of the arbor. Project Goals: The main neuroscientific goal of this project is to understand how multiple local interactions of cytoskeleton components during differentiation define mature dendritic arbor shape and its functional integrative properties, using Drosophila sensory neurons as a model. The technological goal of this project is to develop a novel investigative approach that integrates and extends previously separate approaches from developmental biology & genetics, in vivo confocal imaging & electrophysiology, computer vision, and neuroanatomical modeling. Specific Aims: We propose 3 tightly integrated specific aims. Aim 1: use genetic manipulations and electrophysiological recordings to model the role of cytoskeletal organization and dynamics as a fundamental determinant of emergent dendrite arbor shape and function. Aim 2: Implement advanced 4D multi-parameter imaging protocols and automated algorithms to reconstruct the arbor, and quantify spatial and temporal associations among multiple sub-cellular components. Aim 3: using automated reconstructions & measurements from aim 2, statistically characterize the structural and cytoskeletal features of dendrite arbors, and stochastically simulate the growth and electrotonic properties of anatomically realistic virtual neuronal analogues. The data from aim 3 will feed back novel hypotheses to be tested by a subsequent repetition of the (aim 1 - aim 2 - aim 3) cycle. Approach: We will focus on a single model system - Drosophila dendritic arborization (da) sensory neurons. More specifically, we will investigate class I and class IV da neuron arborization based upon their radically distinct dendritic morphologies (simple vs. complex) and underlying cytoskeletal organizations. We will make fusion constructs of cytoskeleton components with spectrally distinct fluorescent proteins. These will be used in transgenic Drosophila in order to quantitatively measure the distribution of F-actin, microtubules, and microtubule polarity within the dendrite arbor throughout its development in vivo using confocal multi-fluor imaging. The resulting images will be processed by automated quantitative computer vision algorithms that will accurately extract the topology of the dendritic arbor, and it changes over time. We will use the resulting maps in neuroanatomical stochastic simulations to establish the links between the emergent morphometrics of the dendrite and specific cytoskeleton features at various developmental stages. Intellectual Merit: From a neurogenetics perspective, this project will pioneer the use of cytoskeletal features as putative fundamental determinants in statistical neuroanatomical models. These determinants will be linked to morphological determinants. From a computational perspective, this project will advance the state of the art in automated algorithms for delineating neuroanatomy (and its morphological dynamics) by deploying core technologies for large-scale multi-parameter studies, and result in an effective interfacing of automated reconstruction and simulation technologies. With this innovation, model predictions can be tested by molecular biological techniques, and findings of statistical models can be used to inform molecular models of dendrite arbor development. Educational Impact: This project will result in a cross-disciplinary training of post-doctoral fellows, graduate students, undergraduate students and high school interns. It will result in practical insight on ways to conduct cutting-edge systems-level scientific research overcoming disciplinary boundaries and using best-available collaborative tools. The trainees from this program will be uniquely positioned to develop the broader field of imaging-driven integrative systems neurobiology. It will expose minority and K-12 students to a new world of trans-disciplinary research that is indicative of the future. Broader Impacts: The combined body of molecular, imaging, and computational tools and datasets from this research will be disseminated widely, and made available to a broad class of investigators for adoption in the study of other major neuroscience problems. This project will serve as a new model for computationally enabled neuroscience research that achieves a long-desired synergy between the wet lab and computation.

The long-term goal of this proposal is to understand the molecular and physiological bases of cold nociception. Thermosensory nociception is a specialized form of somatosensation essential to the survival of all metazoans. Thermosensory nociception alerts the organism to potential environmental dangers coupled with pain sensation thereby serving as a protective mechanism for driving adaptive behavioral responses to safeguard against incipient damage. Despite this importance, the fundamental molecular and biophysical bases of cold nociception remain poorly understood. Molecularly, transient receptor potential channels (i.e. thermoTRPs) play critical roles in thermosensation, however, relatively less is known regarding how thermoTRPs mechanistically function in regulating noxious cold detection. Neurologically, acute and chronic pain may manifest as altered thermosensory nociception whereby innocuous thermal stimuli erroneously engage nociceptive circuitry leading to neuropathic pain. Cold hypersensitivity is associated with multiple sclerosis, fibromyalgia, stroke, and chemotherapy-induced neuropathy resulting in neuropathic pain, however the mechanisms underlying cold sensitization are largely unknown. Here, we will investigate a fundamental problem of how multimodal sensory neurons discriminately detect noxious cold stimuli to elicit nocict9ptive behavior using Drosophila as a model system in combination with bi- directionally linked neurogenetic, neurogenomic, cellular imaging, electrophysiological, behavioral, computational modeling, and bifurcation analyses. We aim to uncover molecular and biophysical bases for cold-evoked nociceptive stimulus coding, including the functional properties of thermoTRPs and Ca2· signaling dynamics in this process. The project aims and outcomes of this research will significantly advance our knowledge of cold nociception by addressing three open questions: (1) What are the molecular and biophysical bases of cold nociceptive stimulus coding? (2) How do multimodal nociceptive neurons discriminately detect noxious stimuli (e.g. cold) to drive nocifensive behavior? (3) How do thermoTRPs and Ca2· signaling mechanisms mechanistically function in regulating noxious cold detection? More generally, the bi-directional integration of experimental and computational approaches in a closed- loop investigational strategy is well-suited to transform our understanding of cold nociception by elucidating potentially generalizable mechanisms of cold thermosensory coding, including roles of TRP channels and. Ca2· homeostasis in sensory-evoked neural activity. RELEVANCE (See instructions): The perception of noxious stimuli is often coupled to pain sensation as a protective mechanism, however altered temperature sensation may lead to neuropathic pain (e.g. in multiple sclerosis, fibromyalgia, and stroke) where patients experience pain due to cold hypersensitivity. By uncovering basic mechanisms of noxious cold perception, we develop important insights on neural integration of painful stimuli providing potential routes for understanding and treating neurological disease when this process is disrupted.

Abstract The specification and dynamic modification of subtype specific dendritic architecture not only dictates how distinct classes of neurons form functional connections with other neurons, but also directly influences subtype- specific computational properties. Dendritic form, and by extension function, is chiefly mediated by subcellular organization and dynamics of cytoskeletal components. Thus, identifying molecular factors and cellular processes that regulate subtype specific dendritogenesis is essential to our understanding of the mechanistic links between cytoskeletal organization and neuronal form and function in both health and neuropathologies. Protein homeostasis, or proteostasis, is essential to cellular health and as a surveillance system against neurotoxic aggregates implicated in numerous neurodegenerative disease states. Despite this importance, relatively little is known regarding the normal developmental roles of proteostasis regulatory pathways in driving dendritic diversity or subtype-specific cytoskeletal organization. Our work in the previous funding cycle provided the foundations for combining neurogenetic manipulations, in vivo spatio-temporal multichannel imaging and computational techniques for multichannel and time-varying neuronal reconstructions of subtype specific dendritic cytoskeletal architectures. This strategy yielded novel insights into local cytoskeletal control mechanisms regulating dendritic arbor diversity that could not have been solely predicted or quantitatively characterized without the synergy of these approaches. For this next funding cycle, we hypothesize that the evolutionarily conserved PP2A phosphatase and TRiC/CCT chaperonin complexes function as essential proteostasis regulators that exert control over the spatiotemporal organization and dynamics of cytoskeletal components underlying subtype-specific dendritic arbor diversity. To investigate this core hypothesis, we propose the following tightly linked aims. First, we will elucidate the mechanistic role(s) of the PP2A phosphatase and TRiC/CCT chaperonin complex in directing subtype specific dendritic arborization. Second, we will identify the functional requirements and putative molecular targets of PP2A and TRiC/CCT in regulating subtype specific dendritic cytoskeletal architecture and dynamics. Third, we will conduct computational studies of dendritic morphology and spatio-temporal cytoskeletal distributions that directly integrates and synergizes with the first two aims thereby generating a closed-loop investigational system. These studies will not only reveal novel molecular mechanisms driving cytoskeletal organization and dynamics that functionally contribute to the emergence of diverse dendritic arbors, but also develop and disseminate neuroinformatic tools and data of broad impact to the neuroscience community.

? DESCRIPTION (provided by applicant): The proposed Initiative for Maximizing Student Development (IMSD) at Georgia State University (GSU) offers a highly innovative program that will recruit and retain undergraduate students from groups underrepresented in science who will then pursue careers in biomedical sciences. GSU is an ideal institution for an IMSD program as it combines excellence in biomedical and behavioral sciences with a remarkably diverse undergraduate population as well as with GSU's success in training undergraduates from underrepresented minorities. GSU now graduates more African American students than any other non-profit institution of higher learning in the United States. The goal of the proposed IMSD is to combine our existing strengths in biomedical research with the unique success of GSU in educating underrepresented minorities to make it the premier institution in the nation for the education of a highly diverse group of students who will go on to NIH-funded research careers. The objectives of this IMSD program are: (1) to engage high-potential undergraduates from underrepresented groups, recruited from Neuroscience, Biology, Chemistry, and Psychology, in a two-year research immersion and integration program, which includes research during summer as well as academic year activities that will integrate regular coursework with research activities (research semester); (2) to promote survival skills for research careers through an intensive series of professional development workshops and courses in which students will hone their skills in critical thinking, scientific communication, research career planning, and ethics; (3) to create a highly supportive academic and social environment to launch graduate careers in biomedical science by providing instructors with skills to train students from underrepresented groups and help them get entry into PhD programs and by fostering connections with other partner T32 and PREP institutions; and (4) to evaluate progress and identify program, mentor, and trainee dispositions that predict success in biomedical research careers, which we will then use to institutionalize successful program elements beyond the grant period at GSU and, by disseminating and publishing the results of our evaluation, at other institutions.