Peter F. Hitchcock - US grants

dendrites; synapses; retina;

The long term objectives of the work proposed here are to contribute to our understanding of the mechanisms that underlie the development of the eye and brain. The immediate aims of this proposal are: 1) describe the contacts made by the dendritic growth cones of ganglion cells within the nascent inner plexiform layer; 2) determine whether or not the temporal differences in the electrical activity of afferent synapses is a guidance cue for dendritic growth 3) characterize the degree to which locally regenerated retina resembles the surrounding, undamaged retina; and 4) experimentally test whether or not the undamaged retina serves as a template upon which a local patch of retina is regenerated. These aims will be achieved by using electron microscopy, light microscopy, intracellular injections of individual retinal neurons with markers for light and electron microscopy, and immunocytochemistry. It is expected that the studies described herein will provide information that will eventually be useful for devising strategies for treating diseases of, or damage to the central nervous system.

The long term objectives of the work proposed here are to contribute to our understanding of the mechanisms that underlie the development of the eye and brain. The immediate aims of this proposal are: 1) describe the contacts made by the dendritic growth cones of ganglion cells within the nascent inner plexiform layer; 2) determine whether or not the temporal differences in the electrical activity of afferent synapses is a guidance cue for dendritic growth 3) characterize the degree to which locally regenerated retina resembles the surrounding, undamaged retina; and 4) experimentally test whether or not the undamaged retina serves as a template upon which a local patch of retina is regenerated. These aims will be achieved by using electron microscopy, light microscopy, intracellular injections of individual retinal neurons with markers for light and electron microscopy, and immunocytochemistry. It is expected that the studies described herein will provide information that will eventually be useful for devising strategies for treating diseases of, or damage to the central nervous system.

Normal function of the vertebrate nervous system depends on a precise pattern of distribution of the different cell types. For example, cone photoreceptors, which constitute a sheet of light-sensitive neurons in the retina of the eye, come in three main types, each type maximally sensitive to light of a certain color, red, green or blue. The different cone-cell types are arranged in a regular, repeating lattice or mosaic pattern. The developmental mechanisms that build the lattice, i.e. that control the spacing and determine the fate of the presumptive cone cells, are not well understood. Most biologists agree that the immature cells send signals to their neighbors that determine the fate of the neighboring cells. To begin to understand this process, the earliest stages in the development of the cone mosaic will be examined. Molecular markers will used to identify the early developing cone types. The hypothesis to be tested is that one cone type acts as the "founder" to establish a blueprint of the mosaic pattern, and the other cone types develop later, filling in the pattern. This study will give new insight into how the distribution of different neuron types are established and will provide new information about the way cells communicate with each other during development.

The long term objectives of the work proposed here are to contribute to our understanding of the mechanisms that underlie the development of the eye and brain. The immediate aims of this proposal are: 1) describe the contacts made by the dendritic growth cones of ganglion cells within the nascent inner plexiform layer; 2) determine whether or not the temporal differences in the electrical activity of afferent synapses is a guidance cue for dendritic growth 3) characterize the degree to which locally regenerated retina resembles the surrounding, undamaged retina; and 4) experimentally test whether or not the undamaged retina serves as a template upon which a local patch of retina is regenerated. These aims will be achieved by using electron microscopy, light microscopy, intracellular injections of individual retinal neurons with markers for light and electron microscopy, and immunocytochemistry. It is expected that the studies described herein will provide information that will eventually be useful for devising strategies for treating diseases of, or damage to the central nervous system.

DESCRIPTION: (Adapted from the applicant's abstract): A hallmark of the adult human nervous system is that damage is permanent. Neurons that die as a result of trauma or disease are never naturally replaced. In marked contrast, an injury to the retina of an adult teleost fish stimulates neuronal regeneration and recovery of function. An improved understanding of the cellular and molecular events that underlie the ability of this animal to functionally repair this part of its central nervous system may yield new strategies or applications for repairing the nervous system in man. The long-term objectives of this research project are to identify the molecules and cells that are critical for neuronal regeneration and fully reconstruct the cellular and molecular cascade of events that are initiated when the teleost retina is injured. The investigators propose to undertake four specific aims that will: (1) test the general hypothesis that in teleosts, retinal regeneration recapitulates retinal ontogeny; (2) identify the cellular patterns of expression of the insulin receptor and the insulin-like growth factor system (IGF-I; IGF-IR and IGFBP-2); (3) test the hypothesis that the IGF-I system is involved in the signaling pathways that initiate regeneration; and (4) characterize the population of slowly-dividing neuronal progenitors that were recently discovered to be present in the differentiated retina, and test whether or not these cells participate in the regenerative response. Both normal and injured/regenerating retinas will be studied utilizing a variety of cellular and molecular techniques. If the hypotheses are correct, then the investigators expect to identify important molecular mechanisms that regulate both neuronal development and regeneration in a vertebrate retina.

tissue /cell preparation; image processing; morphology; biomedical facility; brain cell; eye; light microscopy; vision; photography; electron microscopy; bioimaging /biomedical imaging;

nervous system regeneration; astrocytes; retina; vascular endothelium; angiogenesis; cell cell interaction; vascular endothelial growth factors; cell proliferation; genetic manipulation; platelet derived growth factor; developmental neurobiology; in situ hybridization; polymerase chain reaction; genetically modified animals; laboratory mouse;

protein tyrosine kinase; nervous system regeneration; gene expression; retina; regulatory gene; central nervous system; developmental genetics; Osteichthyes; tissue /cell culture;

PROJECT SUMMARY/ABSTRACT This application from the University of Michigan builds upon a long-standing tradition of training graduate students and postdoctoral fellows for careers in vision research. The centerpiece of our current training efforts is the Vision Research Training Program (VRTP), which is now in its 15th year. The goals of the VRTP are to provide breadth in research training and professional development to keep pace with the opportunities for careers in vision research. The VRTP will (1) recruit and support candidate-level predoctoral fellows as they undertake and complete their thesis research, (2) recruit and support postdoctoral fellows as they begin independent research careers, (3) provide a formal structured education, (4) and require trainees to participate in a program of professional development specific to vision research. Members of the Training Faculty are experienced mentors, who are well-funded, productive scientists that utilize the eye and visual system as models for basic and translational studies. The PI/PD is Dr. Peter Hitchcock, Professor of Ophthalmology and Visual Sciences, Professor of Cell and Developmental Biology and Associate Dean for Programs and Initiatives in the Graduate School. The Associate Director is Dr. Kwoon Wong, Associate Professor of Ophthalmology and Visual Sciences and Molecular, Cellular and Developmental Biology. The annual admission and enrollment of training grant-eligible doctoral students justify our request for 3 pre-doctoral positions per year. Similarly, the number of training grant eligible postdoctoral fellows currently in training justify our request for 1 postdoctoral position per year. The University of Michigan is committed to diversity and strives to create academic communities that are representative of the global society. The faculty, students and staff are from all cultural, ethnic, gender, and socioeconomic backgrounds. The University values the contributions of these diverse groups. The VRTP participates fully in initiatives at the University of Michigan to promote diversity in the biomedical sciences workforce. The University of Michigan requires the highest level of research integrity from its faculty, staff and students. All trainees participate in a multi-tiered program that teaches responsibility in the conduct and administration of research.

DESCRIPTION (Adapted From The Abstract Provided By Applicant): This program will provide broad, early-stage training in the neurosciences to pre-doctoral students at the University of Michigan. It will support 8 Ph.D. students in the interdepartmental Neuroscience Program during their first two years in the program. Once students complete the initial training supported by this grant, they carry out doctoral research in the laboratory of one of 85 different faculty members. About half of the faculty hold primary appointments in basic science departments, and the other half hold primary appointments in clinical departments. Thus, there are opportunities to explore a wide range of possible topics in sensory, integrative, developmental and molecular neuroscience. Many students carry out their thesis research in institutes focused specifically on disorders of the brain or sensory systems (the Mental Health Research Institute, the Kresge Hearing Research Institute, the Kellogg Eye Center and the Michigan Alzheimer's Disease Research Center). Others carry out basic neuroscience research in the departments of Biology, Biological Chemistry, Cell and Developmental Biology, Human Genetics, Physiology, Pharmacology and Psychology. Among areas of particular expertise at the University of Michigan that are represented in these departments are cognitive neuroscience, axonal guidance and neuronal differentiation, synaptic function, movement disorders, neuronal signaling by kinases and phosphatases, and the neurobiology of drugs of abuse. The first two years of this program are designed to make sure that all graduates of the program are broadly trained. In the first year, students take an interdisciplinary, year-long course in the Principles of Neuroscience, two intensive laboratory courses, a course focused on the responsible conduct of research and carry out research rotations in at least two different laboratories. During the second year students take elective neuroscience courses, participate in an intensive neuroscience seminar course, complete their lab rotations and begin the initial work on the doctoral thesis.

The Vivarium Module provides support staff and equipment for the care and maintenance of animals used by participating investigators whose laboratories are in the Kellogg Eye Center.

[unreadable] DESCRIPTION (provided by applicant): Diverse cellular phenotypes and functions in metazoans are specified by differential expression of genes. Regulation of quantitatively precise expression of genes in the right cell type and at the fight time is mediated by the combinatorial and synergistic (or antagonistic) action of a limited number of transcription factors. Nrl, first identified by the PI, is a key basic motif-leucine zipper (bZIP) transcription factor, which is now established as a prime mediator of gene regulation in both developing and mature rod photoreceptors. Nrl interacts with Crx and other transcription regulatory proteins to synergistically (or antagonistically) modulate the expression of rhodopsin and many rod-specific genes. Mutations in the human NRL gene are associated with retinal degenerative diseases. We have shown that the deletion of Nrl by gene targeting in mice (Nrl -/-) results in complete lack of rod function and rod-specific gene expression; instead, there is enhanced S-cone function indicating a phenotypic transformation. Using the Nrl-promoter to drive enhanced green fluorescent protein (EGFP) in transgenic mice, we have demonstrated that Nrl expression during development corresponds to the genesis of rods. Continued expression of Nrl in mature rods suggests that it also plays a major role in maintaining appropriate expression of genes required for rod function. Our studies have identified Nr2e3 (photoreceptor specific nuclear receptor, PNR) as a direct transcriptional target of Nrl and revealed that Nr2e3 acts synergistically with Nrl and Crx in regulating rod phototransduction genes. In this renewal application, we propose to decipher the Nrl-mediated transcriptional regulatory network(s) in developing and mature rod photoreceptors. The specific aims of the project are as follows: Specific Aim 1: We will identify the genes that are directly regulated by Nrl ("direct targets") using a comprehensive strategy, involving microarray analysis, chromatin immunoprecipitation (CHIP) and promoter activity assays. Specific Aim 2: We will identify transcriptional regulatory proteins that interact with Nrl during early and late stages of rod development and validate the physiological relevance of selected interactions. Specific Aim 3: We will determine whether Nrl is sufficient to induce rod-specific gene expression and generate functional rods, by expressing Nrl in the Nrl -/- retina at temporally distinct stages. Specific Aim 4: We will delineate the function of Nr2e3, a direct target of Nrl, in developing and mature rod photoreceptors. Specific Aim 5: We will define the molecular mechanism(s) that underlie the pathogenesis of retinopathies, caused by mutations in the human NRL and NR2E3 genes. Since mutations in Nrl, its interacting proteins, and their target genes result in retinopathies, it is expected that our studies will reveal significant new insights into retinal diseases. A better understanding of transcriptional regulatory pathways may allow us to experimentally manipulate the expression of specific target gene(s) to correct a disease phenotype. [unreadable] [unreadable] [unreadable]

Project Summary/Abstract Injuries or diseases that kill retinal neurons and receptors block vision at it source. The resulting blindness is permanent. The inability to repair in the retina is a hallmark of the human central nervous system; neurons that die are not replaced and functions that are lost are not recovered. This bleak outcome is a driving force for research on neural stem cells and the field of regeneration biology. The long-term objective of this project is to elucidate the mechanisms that regulate the birth, death and regeneration of neurons and photoreceptors in the vertebrate retina. This program of research utilizes the zebrafish, because the fish retina is the only vertebrate CNS tissue where intrinsic stem cells can regenerate a single neuronal type that integrates into an existing circuit or can regenerate all cell types that completely restore the original tissue. Thus, investigating stem cell-based neuronal and photoreceptor regeneration in the teleost retina will advance our knowledge of mechanisms that govern the ability of intrinsic stem and progenitor cells to restore neural circuits in the injured brain. The resulting knowledge will guide the therpeutic use of transplanted stem and progenitor cells to treat retinal injuries, blindness and disease. Three Specific Aims are proposed, each directed toward revealing mechanisms that regulate the genesis and regeneration of photoreceptors. Specific Aim 1 will investigate the function of the bHLH transcription factor, NeuroD, in both the developing and regenerating retina. Specific Aim 2 will test the hypothesis that microglia play a neurogenic role during photoreceptor regeneration. Specific aim 3 will test the hypothesis that the soluble growth factor, midkine-a, controls aspects of retinal development in larval zebrafish and photoreceptor regeneration in adults. Together these specific aims represent a focused and integrated research program to test specific hypotheses of the biology and regulation of retinal development and neuronal regeneration in the vertebrate nervous system.

DESCRIPTION (provided by applicant): This application from the University of Michigan builds upon a long-standing tradition of training graduate students and postdoctoral fellows for careers in vision research. The centerpiece of our current training efforts is the Vision Research Training Program (VRTP), which is now entering its tenth year. This formal program of education and training (1) recruits and supports candidate-level predoctoral fellows as they complete their thesis research, (2) recruits and supports postdoctoral fellows as they begin independent research careers, (3) provides a comprehensive overview of vision research through the biennial course, Fundamental Issues in Vision Research (Ophthalmology 733), (4) hosts a formal seminar series, Vision Lunch (5) provides support for trainees to attend national scientific meetings and (6) directs trainees to events and programs at the University of Michigan focused on career development. The goals of the VRTP are to provide breadth in research training to keep pace with the opportunities for independent research in vision science. Members of the Training Faculty are experienced mentors, who are well-funded, productive scientists that utilize the eye and visual system as models for basic and translational studies. The PI/PD is Dr. Peter Hitchcock, Professor of Ophthalmology and Visual Sciences and Professor of Cell and Developmental Biology. The University of Michigan is committed to diversity and strives to create academic communities that are representative of the global society. The faculty, students and staff on our campus are from all cultural, ethnic, gender, and socioeconomic backgrounds. The University values the contributions of these diverse groups, not only as they impact our scientists and teachers in- training, but also for the real potential tat their work can improve the health and welfare of traditionally underrepresented and disadvantaged populations around the world. The VRTP participates fully in initiatives at the University of Michigan to promote diversity in the biomedical sciences workforce. The University of Michigan requires the highest level of research integrity from its faculty, staff and students. ll trainees participate in a multi-tiered program that teaches responsibility in the conduct and administration of research. This program addresses each of the five essential review criteria for training in the responsible conduct of research. As evidenced by the productivity and success of our trainees, the research training accomplishments of VRTP have been outstanding.

Project Summary/Abstract Injuries or diseases that kill retinal neurons or photoreceptors block vision at it source. The inability to repair the retina is a hallmark of the human nervous system; neurons that die are not replaced and functions that are lost are not recovered. This bleak outcome is a driving force for research on neural stem cells and the field of regeneration biology. The long-term objective of this project is to elucidate the molecular mechanisms that regulate the birth, death and regeneration of neurons and photoreceptors in the vertebrate retina. This program of research utilizes the zebrafish retina, the only vertebrate CNS tissue in which intrinsic stem cells can regenerate a single neuronal type that integrates into existing synaptic circuits or regenerate all cell types that completely restore the original tissue. Investigating stem cell-based developmental and regenerative neurogenesis will advance our knowledge of the mechanisms that govern retinal stem and progenitor cells and will guide the therpeutic use of stem and progenitor cells to treat retinal injuries, blindness and disease. Three Specific Aims are proposed, each directed toward revealing mechanisms that regulate the genesis and regeneration of retinal neurons and photoreceptors. Specific Aim 1 will test the hypothesis that photoreceptor genesis is governed through post-transcriptional regulation of NeuroD by the the microRNA, miR-18a. Specific Aim 2 will determine the combined and independent functions of the Midkine paralogs during retinal development and photoreceptor regeneration. Specific aim 3 will test the hypothesis that the matrix metalloproteinase, MMP9, is induced in Müller glia by TNF-?, and together these molecules are components of an acute inflammatory response that governs photoreceptor regeneration. Together these specific aims represent a focused and integrated research program to test specific hypotheses about the biology and regulation of developmental neurogenesis and adult photoreceptor regeneration in the vertebrate retina. This program of research will expand our knowledge of photoreceptor death and regeneration and the molecular mechanisms that regulate retinal stem and progenitor cells. These studies also will have implications for the NEI Audacious Goals Initiatives.

Project Summary For twenty years there has been ongoing discussion of the changing nature of careers for bioscientists, whether we are training too many Ph.D.s, and whether we are preparing our students optimally for future careers. Discussions of these topic have intensified in the last few years, largely dominated by a few strong voices at elite levels of academic science. The purpose of a 2015 meeting at the University of Michigan was to bring together representatives of graduate and postdoctoral training at members of research-intensive universities and medical schools with representatives from federal funding agencies, the private sector, trainees, and foundations to openly discuss many of the proposals that have been made (rather than rehash the problems) and look for the best ways forward. The meeting produced both broader input to existing ideas and new lines of thought that could be further developed by the stakeholders. We now propose a collaborative effort between the University of Colorado and the University of Michigan to host a follow-up meeting in Denver on the Anschutz Medical Campus in June, 2017. The two year intervening period has allowed continuing discussions among the NIH, NSF, private foundations, and selected interested parties, as well as the implementation of innovative training models funded by the stakeholders (e.g. NIH's BEST programs and NSF's NRT programs). The proposed 2017 meeting is intended to catalyze a broad collaboration of stakeholders to create a set of integrated and strategic proposals for the future, and that these will undergo detailed discussion by a broad cross-section educational institutions, trainees, and potential employers at the meeting to create consensus on effective practices and potential policy shifts. The conference organizers and session chairs will collaborate to produce a publication to disseminate these meeting outcomes. It is anticipated that 400-500 people will participate in these discussions and will provide substantial momentum to move forward with adjustments to institutional and national policies. The University of Colorado Denver and the University of Michigan are investing substantial funds, personnel, and space resources. Support from NIH is sought to fund travel grants to allow attendance by graduate students and postdoctoral scholars who engaged in these discussions regionally and nationally, as well as other colleagues who could contribute expertise but would not otherwise have the resources to attend.

DESCRIPTION (provided by applicant): This application from the University of Michigan builds upon a long-standing tradition of training graduate students and postdoctoral fellows for careers in vision research. The centerpiece of our current training efforts is the Vision Research Training Program (VRTP), which is now entering its tenth year. This formal program of education and training (1) recruits and supports candidate-level predoctoral fellows as they complete their thesis research, (2) recruits and supports postdoctoral fellows as they begin independent research careers, (3) provides a comprehensive overview of vision research through the biennial course, Fundamental Issues in Vision Research (Ophthalmology 733), (4) hosts a formal seminar series, Vision Lunch (5) provides support for trainees to attend national scientific meetings and (6) directs trainees to events and programs at the University of Michigan focused on career development. The goals of the VRTP are to provide breadth in research training to keep pace with the opportunities for independent research in vision science. Members of the Training Faculty are experienced mentors, who are well-funded, productive scientists that utilize the eye and visual system as models for basic and translational studies. The PI/PD is Dr. Peter Hitchcock, Professor of Ophthalmology and Visual Sciences and Professor of Cell and Developmental Biology. The University of Michigan is committed to diversity and strives to create academic communities that are representative of the global society. The faculty, students and staff on our campus are from all cultural, ethnic, gender, and socioeconomic backgrounds. The University values the contributions of these diverse groups, not only as they impact our scientists and teachers in- training, but also for the real potential tat their work can improve the health and welfare of traditionally underrepresented and disadvantaged populations around the world. The VRTP participates fully in initiatives at the University of Michigan to promote diversity in the biomedical sciences workforce. The University of Michigan requires the highest level of research integrity from its faculty, staff and students. ll trainees participate in a multi-tiered program that teaches responsibility in the conduct and administration of research. This program addresses each of the five essential review criteria for training in the responsible conduct of research. As evidenced by the productivity and success of our trainees, the research training accomplishments of VRTP have been outstanding.

Rumination may promote ineffective behavior that ultimately harms mental health, although the mechanisms are unclear. Recent research suggests rumination, which is defined as dwelling on the possible significance and implications of one?s distress and problems, may promote ineffective behavior by disrupting reinforcement learning (RL). RL refers to the suite of processes that enable adjusting expectations, when an experience differs from expected, and then taking appropriate action. RL is a ubiquitous process that is necessary for effective behavior. Hence, rumination?s disruption of RL may represent an upstream process that helps explain rumination?s link to various ineffective behaviors. This raises the prospect of intervening to prevent rumination from disrupting RL, thereby making behavior more effective, and ultimately improving mental health. However, the recent research showing rumination disrupts RL is currently unclear on if rumination disrupts the RL mechanisms of learning (adjusting expectations) or choice (taking appropriate action). Different mechanisms would have distinct translational implications, hence clarity on the specific mechanisms is critically needed. Therefore, the objectives of this project are to dissociate rumination?s effects on the learning and choice components of RL, and to apply computational modeling and electroencephalography (EEG) to identify the specific mechanisms of these components it impairs. The central hypotheses are that rumination disrupts RL by impairing the following RL mechanisms: guidance by working memory that accelerates learning, long-term punishment learning, and cognitive control that enables effective choice. To test these hypotheses, the within- subject effects of ruminative (RUM) vs. neutral (NEU) thinking on mechanisms that guide learning (Aim 1) and choice (Aim 2) in RL will be investigated. To ensure generalization to clinical rumination, all participants (N=60) in this single-visit experimental study will meet criteria for Major Depressive Disorder or Generalized Anxiety Disorder. To ensure feasibility, recruitment will be embedded in a large-scale external project that conducts diagnostic interviews. Aim 1 will examine if RUM (vs. NEU) leads to slower acquisition of contingencies by disrupting working memory, and causes working memory to represent punishment experiences more strongly during initial learning, thereby disrupting long-term punishment learning. Aim 2 will examine if RUM (vs. NEU) disrupts cognitive control in a task where Pavlovian (instinctual) and instrumental (goal-directed) impulses conflict, and cognitive control is needed to override the former. These aims are integrated with a training plan to broaden and deepen the applicant?s background in computational neuroscience, to enable him to acquire specialized skills in this area (hierarchical Bayesian computational modeling and EEG), and to facilitate his development of a programmatic and competitive translational research program. This training is essential to the applicant?s transition to an independent, productive research career in the NIMH priority area of computational psychiatry.