Rosalind A. Segal - US grants

DESCRIPTION: Purkinje cells are the sole output from the cerebellar cortex, and so diseases that alter Purkinje cell develop and function have devastating effects on balance and coordination. My colleagues and I have demonstrated that the neurotrophin, BDNF, is necessary for normal Purkinje cell development, particularly for dendritic arborization. In the proposed research we will identify the cells that synthesize BDNF to regulate Purkinje cell development and determine whether the p75 receptor plays a role in this regulation. We will investigate further the nature of the biological effects of BDNF on Purkinje cells, to determine whether BDNF regulates Purkinje cell synapses. Specific aim 1 is to test the hypothesis that BDNF synthesized and released by granule cell acts in an anterograde fashion to regulated Purkinje cell development; we will also examine the alternative possibilities that BDNF is instead involved in the interactions of climbing fiber or deep cerebellar nuclei neurons with Purkinje cells. We will use genetic or mechanical means to remove granule cells in vivo or remove afferent connections to the cerebellar cortex, and determine whether Purkinje still accumulate BDNF and undergo Trk phosphorylation. We will use organotypic and dissociated cell cultures to determine whether BDNF is required for granule cells to regulate Purkinje cell dendritic arborization. Specific aim 2 is to test the hypothesis that both TrkB and the p75 receptor mediate the effects of BDNF in developing Purkinje cells. While our previous work has demonstrated that activation of the TrkB receptor is a critical component in the Purkinje cell response to BDNF, others have shown that antibodies to p75 perturb Purkinje cell dendritic growth and survival, suggesting that both receptors may participate in the BDNF response. We will analyze Purkinje cell morphology, cerebellar foliation, Trk phosphorylation and BDNF uptake in p75/BDNF compound mutant mice. These experiments will indicate signal transduction pathways critical for regulating Purkinje cell development. Specific aim 3 is to test the hypothesis that BDNF is involve din the formation and maturation of granule cell-Purkinje cell synapses. We will use structural, biochemical and electrophysiologic approaches to compare parallel fiber: Purkinje cell synapses in wild type and BDNF-/- mice, and to evaluate acute and chronic effects of BDNF on Purkinje cell synapses. These studies will identify extracellular factors that regulate Purkinje cell development, and could provide the basis for therapeutic applications of neurotrophic factors on drugs in treating cerebellar diseases.

Target derived neurotrophins regulate neuronal survival and function. Little is known about the mechanisms by which neurotrophins regulate events in the nerve cell body when presented to nerve ending that may be as far as a meter away. Previously, we have shown that neurotrophin receptors (Trks) themselves serve as rapid retrograde signal carriers in rat sciatic nerve axons. The research described here builds upon this work. We have three specific aims: Aim one is to learn how Trks function as retrograde signal carriers. Our preliminary data suggest that a specialized vesicular transport process is utilized to convey Trk signal generating particles in the retrograde direction. To test this hypothesis, activation state-specific antibodies will be used as immuno-electron microscopy reagents and as immunoaffinity reagents to localize and isolate the Trk signal carrying complex in rat sciatic nerve axons. Temperature sensitive mutations of dynamin will be expressed in compartmented cultures of DRG neurons to evaluate the role of receptor internalization in retrograde signaling. Retrograde movement of activated Trks will be visualized in living DRG neurons using Green Fluorescent Protein tags. Aim two is to learn how P-Trk signal arriving at the nerve cell body is converted to a nuclear signal. Our preliminary data suggest that Trk signal arriving from a remote source of stimulation (the nerve ending) is processed by ERK isoforms distinct from the ones that process Trk signal from a local source (the cell body). We will use genetic and biochemical approaches to identify ERKs that are activated by retrograde signal and evaluate their role(s) in nuclear responses. Aim three opens a new avenue of inquiry. Our preliminary data suggest that PI3 kinase activity is required for nuclear responses to remote, but not local, neurotrophin stimulation. PI3 kinases are known to regulate membrane trafficking. In addition, phospholipid products of PI3 kinases serve as activators for serine/threonine protein kinases such as Akt. Genetic and biochemical approaches will be used to test the hypothesis that one or both of these PI3 kinase functions are required for retrograde signaling through long axons. These studies will shed light on the molecular basis of neurodegenerative diseases, and provide guidelines for rational drug design and delivery in treating such disorders.

DESCRIPTION (provided by applicant): During development of the nervous system, precursor cells divide in specialized proliferative zones, then migrate away from these zones and differentiate. The mechanisms that allow coordinated cell cycle exit and directed migration can readily be examined in the developing cerebellum. In studies supported by this grant, my colleagues and I identified two extracellular factors that regulate migration of cerebellar precursors: stromal cell derived factor (SDF-1 alpha) and brain derived neurotrophic factor (BDNF). We demonstrated that mice with targeted gene deletions of either SDF-1alpha or of BDNF exhibit both aberrant migration and proliferation of neuronal precursors in the cerebellum. In the proposed studies we will define the mechanisms whereby these factors stimulate migration, and how they coordinately influence proliferation. 1. The first specific aim is to determine the mechanism by which BDNF promotes granule cell migration. We will use genetic and pharmacologic tools to identify the signal transduction pathways required for directed migration, focusing on the possibility that TrkB activity stimulates PI3 kinase to promote cell migration. 2. The second specific aim is to test the hypothesis that a gradient of BDNF causes redistribution of TrkB receptors to amplify the gradient and provide a direction for chemotaxis. Preliminary data indicate that BDNF induces an asymmetric distribution of the BDNF receptor, TrkB, with accumulation of receptors at the leading edge. We will test the hypothesis that redistribution of TrkB receptors allows amplification of the gradient, determine whether TrkB redistribution reflects movement of surface receptors or the addition of new receptors to the surface, and determine whether receptor redistribution is needed for chemotaxis. 3. The third specific aim is to test the hypothesis that SDF-1alpha functions as a spatially restricted competence factor that limits SHH-induced proliferation to the EGL. We will investigate the possibility that SHH and SDF function as competence and progression factors for precursor proliferation, and identify transcriptional targets that depend on the synergy of these two factors Taken together, these studies will identify mechanisms that regulate the directed migration of granule cells, and define the relationship between migration and proliferation. Since unregulated migration and proliferation are the hallmarks of malignancy, these studies will provide a basis for understanding and treating brain tumors.

DESCRIPTION (provided by applicant): Recent data that mutations in genes encoding dynein components cause Amyotrophic Lateral Sclerosis indicate that dynein function and retrograde axonal transport are required for motor neuron survival. However, the critical molecules transported by dynein motors are not known. Neurotrophins, peptide growth factors released by targets of innervation, initiate a survival signal that must be propagated through the axon. Cell death in Amyotrophic Lateral Sclerosis (ALS), then, may result from impaired transport of a long-range neurotrophin survival signal. In the proposed studies, we will test the hypothesis that long distance signaling by neurotrophins requires intracellular transport processes that are aberrant in ALS. In support of this hypothesis, we have found that dynein function within axons is required for survival of neurons that depend on target-derived neurotrophic factors. In the proposed experiments we will investigate the nature of the intracellular trafficking responsible for retrograde transport of Trk receptors. We will determine whether mutations that cause ALS in humans impede neurotrophin retrograde signaling. These experiments will provide insight into the pathogenesis of ALS, and identify potential therapeutic targets. We have three specific aims: Aim 1: To test the hypothesis that neurodegeneration in ALS results from the loss of long range neurotrophin signaling. To determine whether genetic changes that predispose to motor neuron degeneration impair retrograde neurotrophin signaling, we will use SOD-1 mice (transgenics expressing the SOD-1 G93A mutation). We will ask whether retrograde neurotrophin signaling is impaired in the neurons of these animals fated to develop ALS. Aim 2: To determine whether Alsin, the product of the ALS2 gene at 2q33-35, is required for vesicular trafficking of Trk receptors and for retrograde signaling by neurotrophins. Mutations in ALS2 have been implicated in a large number of familial motor neuron disorders. Alsin, the protein encoded by ALS2, is a guanine nucleotide exchange factor for the endosomal protein Rab5, and facilitates endocytic sorting in the nervous system. We will determine whether impaired Alsin function results in a loss of neurotrophin survival signals. Aim 3: To identify the nature of the dynein motors that transport long range survival signals, and whether these are assembled in response to neurotrophin stimulation. The composition of dynein motor complexes varies based on the cargo that is transported. We will take both a candidate approach and a proteomic approach to identify components of the motor that transports activated Trks, and whether these motors are regulated by neurotrophins. Taken together, these studies will determine how defects in axonal transport cause selective degeneration of motor neurons. The identification of survival signals that depend on transport, and the transport mechanisms, will lead to new therapeutic approaches to ALS.

DESCRIPTION (provided by applicant): The neurodegenerative disease amyotrophic lateral sclerosis (ALS) can be caused by mutations in components of the intracellular motor protein dynein [1]. What, then, are the critical molecules transported by dyneins that are essential for neuronal survival? My colleagues and I have shown that dynein-based transport is required for survival of neurons that depend on target-derived neurotrophic factors. We have shown that this reflects the role of axonal dynein in mediating transport of activated neurotrophin receptors (Trks) within large signaling endosomes. Therefore, we hypothesize that ALS may result from deficiencies in long-range neurotrophic factor signaling, and that restoring these retrograde signals will provide an effective therapeutic approach to the disease. We have identified a set of genes that are preferentially induced by neurotrophin stimulation of distal axons (retrograde signals) rather than by stimulation of cell bodies . (anterograde signals), a set that we designate as retrograde response genes. The retrograde response gene set contains several members that are likely to protect cells from progressive neurodegeneration, including the anti-apoptotic gene bclw and the survival-promoting factor IGF-1. In the proposed studies we will build on our identification of spatially selective neurotrophin responses to determine the mechanisms and functions of specialized retrograde signals. We have three aims. 1. To determine the mechanisms responsible for induction of retrograde response genes by target-derived , neurotrophins, using both motor and sensory neurons grown in compartmented cultures. 2. To determine the functions of retrograde response genes in developing and mature neurons. We will focus on bclw, a poorly understood pro-survival bc!2 family member that is preferentially expressed in the mature nervous system, and can protect diverse neurons from apoptotic stimuli. 3. To test the hypothesis that mutations in dynein that cause ALS do so by interfering with the signaling pathways that induce bclw and other retrograde response genes. We will determine whether expression of these genes protects motor neurons from progressive degeneration. Understanding the mechanism and significance of long distance survival pathways may lead to new therapeutic approaches for ALS and other devastating neurodegenerative disorders.

Recent studies have focused attention on the role of mitogenic niches in regulating stem cell self?renewal, and have emphasized the importance of proteoglycans in forming such microenvironments. However, more than 50 years after the genetic code was deciphered, we do not know whether sugar chains on proteoglycans encode biologically important information. The potential complexity of such a ?glyco code? is enormous, but little is known about the features of the proteoglycans involved in mitogenic regulation, or the signaling mechanisms required for stem cell renewal. To decipher whether there is a glyco code we will identify proteoglycans that specify stem cell renewal in the mammalian brain. We will rely on a genetic approach to proteoglycan biology and on newly developing innovations in mass spectrometry that allow large scale analysis of the sugar composition of proteoglycans. We will generate mutated growth factors capable of binding to cognate receptors, but unable to bind proteoglycans. We will ascertain whether individual proteoglycans modulate the signaling pathway and the biological response elicited by a growth factor, perhaps by influencing the location, presentation, or oligomerization of the factor. We will begin by focusing on Sonic Hedgehog (Shh), which is mitogenic for stem cells in the cerebellum, cortex, and in diverse cancers. We will identify proteoglycans required for Shh- mediated proliferation using a new assay for mitogenic niches, and we will determine how a glyco code might regulate stem cell propagation. As the work progresses we will extend our studies to define proteoglycan structures that modulate the response of stem cells to additional agents such as EGF or FGF family members. These studies will contribute to the identification of a glyco-code, and will determine mechanisms that maintain ?stemness?. Such studies can lead to enhanced therapies for disorders from Alzheimers disease to cancers to stroke.

Segal, Rosalind A. 2 R01 NS037757-10A2

DESCRIPTION (provided by applicant): For many years, conventional wisdom declared that no protein translation occurs in the axons of mammalian neurons. Instead all proteins needed for axonal functions were supposed to be synthesized in the cell bodies and shipped out to the axons. More recently, accumulating data has provided evidence that many mRNAs are found in axons in the mammalian nervous system. Evidence indicating that local translation in axons may be regulated by electrical activity, neurotrophic factors, and stress, have led to the hypothesis that axonal translation is particularly important for plasticity during learning, or in response to environmental stressors. In support of this idea, defects in local translation have been linked to Fragile X syndrome, which causes cognitive problems and autistic behaviors, and paraneoplastic disorders, which cause encephalitis in some patients with lung cancers. However, the mechanisms regulating local translation, and why defects in local translation lead to neuro-psychiatric dysfunction, are not understood. In part, this reflects the inadequacy of current approaches for analyzing local protein synthesis or identifying biological functions that rely on regulated local translation. To address this problem we initiated a collaboration with Michael Lin. Michael recently developed a technique called timeSTAMP, to identify proteins that are translated at a particular time period (4). We are working with Michael to modify this approach so it can be used for spaceSTAMP, to tag proteins translated in a particular location, and follow them over time and space. The goal of this proposal is develop the spaceSTAMP approach and use it to ask: Is there regulated local translation in axons? Are such locally translated proteins functionally important? Are locally translated proteins restricted to the axonal compartment, or do they facilitate communication between axonal terminals and remote portions of the neuronal cell body? These studies will develop and test a spaceSTAMP technique that allows one to tag proteins synthesized in axons, and to follow these components in time and space. This approach can then be used by the scientific community to solve problems such as the functional importance of the fragile X gene product, and ways in which activity or neurotrophin regulated translation contribute to neuro-psychiatric disorders.

DESCRIPTION (provided by applicant): The Dana-Farber/Harvard Cancer Center (DF/HCC) is a Harvard-wide, NCI-designated Comprehensive Cancer Center. This training grant is the principal instrument of basic science training at DF/HCC. Our broad goal is to teach young scientists at the predoctoral and postdoctoral levels how to apply emerging technology in genomics and proteomics to fundamental problems in cell division, cell differentiation or cell death (the three D's of cancer cell biology) that underlie human neoplastic disease. We propose to appoint 5 predoctoral and 12 postdoctoral scientists per year. Our pre-doctoral trainees will be selected from a pool of students who have enrolled in a newly created Cancer Biology Track in the Harvard Biological and Biomedical Sciences. These students will be appointed after their second year of study when they have completed laboratory rotations and chosen one of our mentors as their thesis advisor. The postdoctoral appointments will be for recent recipients of the Ph.D. or M.D./Ph.D. degrees. We are especially selective with our postdoctoral appointments. By multiple metrics the postdoctoral trainees appointed to our program in years past have been every bit as successful as postdoctoral fellows supported by individual awards from other funders, such as NRSA, ACS and Damon-Runyon. Funding for Clinical training is excluded from this program. Laboratory training in cancer research is complemented by a didactic program that prepares our students to exploit a broad range of job opportunities in settings ranging from the small liberal arts college, to academic medical research institutes and the biotechnology industry. Programs provided by the Postdoctoral and Graduate Student Affairs Office at Dana-Farber, which are available for all our trainees, enhance trainee cohesiveness and program identity. The didactic and one-one-one training is complemented by retreats and poster sessions that draw students, postdocs and their mentors from all major components of DF/HCC. On a tactical level, each Department/Division within DF/HCC has journal clubs and seminar programs that provide a sense of local community.

The long-term goal of this program is to improve the standard of care for pediatric astrocytomas - the most common brain cancers in children. Towards this end, we will improve our understanding of astrocytoma biology and develop new diagnostic, prognostic and therapeutic tools for these tumors. The significance of the work is that primary cancers of the central nervous system have now surpassed leukemia as the leading cause of cancer-related death in children. Project 1 draws upon recent observations of activating mutations in BRAF in ~50% of pediatric low grade astrocytomas and addresses three unresolved questions. William Hahn, MD/PhD and Jean Zhao, PhD will study: (i) what are the driving mutations in the ~50% of tumors wild type for BRAF, (ii) what are the mutations that co-occur with BRAF and (iii) what other intracellular kinases are co-activated with BRAF? An innovative feature of this project is recently developed methods for genetic profiling of formaldehyde-fixed, paraffin-embedded samples. These paraffin-friendly technologies greatly expand the available samples of these pediatric tumors. Project 2 addresses the bHLH transcription factor Olig2, with a chemical focus. Olig2 is a strong candidate for targeted therapy of pediatric astrocytomas. However, transcription factors are generally considered to be unattractive targets for drug development because their interactions with DNA involve large and complex surface area contacts. Another generic problem in brain tumor drug development is ensuring delivery beyond the blood/brain barrier. Charles Stiles, PhD and Loren Walensky, MD/PhD propose to develop specific inhibitors of Olig2 with good penetrance properties for the blood/brain barrier. Innovative features of this project are (i) stapled peptide chemistry to create Olig2 antagonists used with (ii) MALDI mass spectrometry imaging technology to address drug penetrance into the interstitial areas of the brain. Project 3 addresses the role of microenvironment in tumor growth. Rosalind Segal, MD/PhD has developed a novel assay for testing the effects of microenvironments on astrocytoma cells. In collaboration with neurosurgeon Liliana Goumerova, MD, she will use tumor cells from pediatric astrocytomas derived from different brain regions to determine whether tumor cells are addicted to the location where they originated, and whether tumor cell niches promote tumor growth, survival, and/or chemoattraction. These studies may lead to new strategies for disrupting the interface between astrocytoma cells and their niches. An innovative feature of this project is a consideration of cilia as signaling organelles that coordinate responses to the microenvironment. The three projects interact with one another and are further unified by economies of scale enabled by an Innovative Neuropathology (INP) core.

The overall objective ofthis project is to develop a new understanding ofthe microenvironment that supports the growth, survival and migration of astrocytoma cells, and thereby identify new therapeutic approaches. In preliminary studies we have shown that human astrocytoma cells do not grow indiscriminately within the brain parenchyma, but prefer to grow in particular microenvironments. In the proposed studies we will use primary cultures from patient samples to determine whether astrocytomas are addicted to the environment of the brain region in which they developed, and to define cellular mechanisms by which the tumor microenvironment promotes tumor maintenance. We have three aims: Aim One: Determine whether pediatric astrocytomas are addicted to the particular location in which they originated. We will use a new slice overiay assay, to determine whether i) forebrain tumors preferenfially grow in the specialized microenvironment of the forebrain; ii) hindbrain tumors preferentially grow in the cerebellum and brainstem areas ofthe hindbrain; iii) BRAF overexpression, a characteristic feature of cerebellar pilocytic astrocytomas, specifically facilitates growth of neural precursor in the cerebellar microenvironment. Aim Two: Define the cellular basis ofthe microenvironment While the microenvironment is cleariy important for tumor growth and maintenance, the reason why tumor cells are preferentially found in particular locations is not understood. We will determine whether i) the microenvironment stimulates tumor proliferafion; ii) the microenvironment selectively promotes tumor cell survival; iii) tumor cells preferentially migrate towards this microenvironment. Aim Three: Determine whether primary cilia coordinate the tumor cell response to its microenvironment. Our preliminary studies indicate that many astrocytoma samples exhibit primary cilia, visualized with antibodies to acetylated tubulin or adenylate cyclase 3. To determine whether primary cilia are signaling organelles that contribute to growth of astrocytomas, we will determine whether i) Primary cilia are preferenfially found on higher grade tumors; ii) Primary cilia point towards the vasculature; iii) components of crifical growth pathways localized to cilia of tumor cells; iv) Primary cilia are required for tumor growth.

instructiors): The broad objective of the Administrative Core is to provide multiple levels of program management support to the project and core investigators. Dr. Segal, as program director, will be responsible for all aspects of program management. To facilitate communication, coordination and planning among the projects and the INP Core, Dr. Segal will be assisted by a scientific program coordinator. Dr. Segal will also be aided by the Cancer Biology Business Office to assist her in the fiscal management of the award. The Administrative Core has three specific aims: Aim one is to provide effective communication, coordination and planning for the program. Dr. Segal and the scientific program coordinator will schedule and facilitate monthly research -in- progress meetings for project and core investigators. The internal advisory board will be invited to this meeting on a semi-annual basis, and the external advisor will be invited to attend yearly, to provide their fresh perspectives and guidance. Following these advisory meetings, Dr. Segal and the program coordinator will synthesize the evaluations and recommendations from the advisory board and distribute them to all project and core investigators. Aim two is to assist the Dr. Segal with fiscal management of the award. This includes processing award notices from the NIH, managing project budgets, and assisting in the preparation of annual progress reports to the NIH. Our Business Office is staffed with grants management specialists who concentrate on either pre-award or post-award aspects of research awards. Aim three is to provide clerical support to Dr. Segal for tracking annual renewals of animal and human protocols, manuscript preparation, traveling and scheduling as it pertains to the program.

DESCRIPTION (provided by applicant): We ask for partial support for the 2011 Neurotrophic Factors Gordon Research Conference. The conference will be held from June 5th through 10th, 2011, at Salve Regina University in Newport, Rhode Island. This conference, held every other year, is viewed as the most important meeting for conveying discoveries of the highest significance in the neurotrophic factor field. It attracts virtually all of the leading scientists studying neuronal growth factors, their mechanisms of action and their roles in development, plasticity and diseases of the nervous system. Moreover, in recent years it has been attended by a significant number of clinicians and scientists in the related fields of developmental neuroscience, and neurological and mental health disorders and by industry representatives whose companies engage in research in neurological and mental health disorders. Over the years, many of the major breakthroughs in the field have been showcased at this meeting. The Conference has played a catalytic role in building the neuroscience community by bringing together both young and established investigators with diverse interests in neurotrophic factors and with expertise in molecular biology, cell biology, signal transduction, synaptic transmission, development, behavior and disease. The overriding objective for the 2011 conference is to enhance communication between scientists working in diverse disciplines to increase their ability to understand neurotrophic factors and to deduce how exploiting their actions may allow us to enhance the lives of individuals with disorders of the nervous system. PUBLIC HEALTH RELEVANCE: The public health relevance of the Conference is its ability to accelerate the transmission of knowledge from fundamental neuroscience to the benefit of patients that suffer with a variety of disorders - Alzheimer's disease, Down syndrome, Parkinson's disease, Huntington's disease, depression, obesity, epilepsy, stroke, and peripheral neuropathies, including those that complicate diabetes and other chronic medical disorders. As such, it fills an important gap in creating and sustaining research with enormous potential benefit to the health and well being of America and the rest of the world. Indeed, we expect this conference to catalyze many new collaborations that address questions important to understanding and treating neurological and mental disorders.

DESCRIPTION (provided by applicant): This proposal is to provide support for junior investigators, post-doctoral fellows and students to attend the 11th Gordon Research Conference (GRC) on Neurotrophic Factors at Salve Regina University, Newport RI from June 2-7, 2013. The meeting will be attended by approximately 150 participants and will feature work on diverse neurotrophic factors, their functions and mechanisms of action. Neurotrophic Factors are extracellular proteins that act on developing or mature neurons to promote cell survival, neural differentiation or synaptic functioning. Defects in neurotrophic factor activities are linke to a broad variety of neuro-psychiatric disorders including autism and cognitive disorders, neurodegenerative diseases, and addictive behaviors. The objectives of this grant are to provide support for a meeting that will: 1. Foster interactions amongst scientists who use a wide variety of approaches to understand the biology of neurotrophic factors in health and in disease. Researchers in this field include cell and molecular biologists, developmental neurobiologists, electrophysiologists and medical scientists. This conference provides a unique forum for investigators using diverse approaches to come together and hear about the most recent advances and methodologies, and for young investigators to learn about the important questions that need to be addressed. 2. Achieve a sense of community among scientists studying neurotrophic factors: The field of neurotrophic factors covers the globe, and multiple aspects of neuroscience. This meeting will feature international scientists in the field, and will highlight many of the accomplished scientists in the field who are women or members of under-represented minorities, and will build a sense of community and cooperation among this group. 3. Provide opportunities for young researchers to present their work and to interact with diverse researchers studying neurotrophic factors: This conference will encourage communication of ideas at the frontiers of science, including a session of hot topics, short talks presented predominantly by students and post-docs. Funds from NIH will be used to enable these young investigators to attend the meeting, and present novel findings in an exciting and collaborative setting.

DESCRIPTION (provided by applicant): We propose to continue a Jointly Sponsored Predoctoral Training Program in Neurosciences that is the major source of support for early year students in the Ph.D. Program in Neurosciences at Harvard University. The goals of this interdepartmental Ph.D. program, established in 1981, are (1) to organize within a single training faculty the neuroscientists at Harvard Medical School, its affiliated hospitals, and Harvard College; and (2) to train research scientists and teachers who are interested in mental health, diseases of the nervous system, and fundamental mechanisms of the brain. The training program is designed to provide trainees with a broad and thorough background in neuroscience and to mentor them in performing original and rigorous research in important areas of neuroscience. In the first 18 months, trainees complete a sequence of core courses ranging from cell and molecular neurobiology to systems neuroscience, as well as collateral courses selected from cell and molecular biology, immunology, statistics, and other subjects appropriate to individual interests. Students rotate through three different laboratories. Following the coursework, laboratory rotations, and a preliminary examination, students begin full time dissertation research. They are also involved in other ongoing training activities including journa clubs, seminars, and data presentation. There are currently 100 graduate students enrolled in the Program in Neuroscience. The total faculty includes 118 members; the 68 faculty who are currently most actively involved in graduate education are training mentors on this proposal. Considerable effort has gone into making this program a highly interactive group with extensive formal and informal contacts between students and faculty. Graduates of this program have a high rate of staying in careers in biomedical research and make substantial contributions to a growing understanding of neuroscience.

? DESCRIPTION (provided by applicant): Provocative question 9 asks: What are the molecular and/or cellular mechanisms that underlie the development of cancer therapy-induced severe adverse sequelae? We respond that untreatable, irreversible neurologic deficits due to cancer chemotherapy constitute a major unmet medical need for cancer patients and cancer survivors. In particular, chemotherapy-induced peripheral neuropathy (CIPN), with deficits in tactile sensation and motor function, affect many cancer patients treated with cytotoxic chemotherapies. These neurologic deficits often limit the doses of chemotherapy that can be used, and cause a major and often permanent impediment to quality of life in cancer patients and survivors. We do not yet understand the underlying mechanism for chemotherapy-induced axonal degeneration, nor do we have any way of effectively treating the resultant neuropathy. Empirical clinical trials for potential therapies have been disappointing. Therefore, to provide a solid basis to develop therapeutics it is imperative to understand the mechanistic process by which chemotherapies cause CIPN. The proposed studies will define the molecular mechanism of CIPN and initiate novel therapeutic approaches for these dire consequences of treatment. Our study plan focuses primarily on neuropathy caused by the chemotherapeutic agent, paclitaxel. Paclitaxel and related compounds are essential for effective chemotherapies for breast, ovarian and other cancers, and the majority of the more than 100,000 patients treated each year with paclitaxel experience symptoms of neuropathy. In preliminary studies using in vivo models and specialized in vitro compartmented cultures to study paclitaxel-induced degeneration of sensory neurons, we have shown that paclitaxel acts directly on axons to initiate degeneration, and that paclitaxel reduces the intracellular level of Bclw (aka Bcl2l2), a protein essential for the lifelong preservation of sensory axons. Strikingly we find that Bclw differs from its closely related family members, Bcl2 and BclxL, in that only Bclw is altered by doses of chemotherapies that cause axonal degeneration, and only Bclw can prevent axonal degeneration caused by paclitaxel. The proposed studies will elucidate the mechanisms whereby paclitaxel and other chemotherapies affect Bclw expression, and how Bclw prevents chemotherapy induced axonal degeneration. Our preliminary studies suggest the exciting possibility that Bclw-mimetics may provide the basis for designing new therapies that limit or reverse neurologic chemotherapy-induced toxicity, much as leucovorin rescue is used to limit and prevent toxicity from methotrexate or as Neulasta is used to alleviate chemotherapy induced bone marrow toxicity.

We propose to continue a Jointly Sponsored Predoctoral Training Program in Neurosciences that is the major source of support for early year students in the Ph.D. Program in Neurosciences at Harvard University. The goals of this interdepartmental Ph.D. program, established in 1981, are (1) to organize the neuroscientists at Harvard Medical School, its affiliated hospitals, and Harvard College into a single training faculty cohort; and (2) to train research scientists and teachers who are interested in mental health, diseases of the nervous system, and fundamental mechanisms of the brain. The training program is designed to provide talented trainees with a broad and thorough background in neuroscience and to mentor them in performing original and rigorous research in important areas of neuroscience. During the first year, students are provided with initial preparation in quantitative approaches to scientific endeavors. This is followed by a year long course, The Discipline of Neuroscience, which provides integrated and rigorous training in concepts central to our understanding of the development, function, and diseases of the nervous system at the levels of cellular signaling, circuit computations, and behavior generation. All students take an additional course in Statistical Approaches to Neuroscience, a neuroanatomy course and two electives. Students also rotate through three different laboratories during the first year. Following the coursework, laboratory rotations, and a preliminary examination, students begin full time, mentored dissertation research. During the program, students are also involved in other ongoing training activities including journal clubs, seminars, retreats, skill workshops, and data presentation. There are currently 102 graduate students enrolled in the Program in Neuroscience; this grant supports 14 students in the first or second year of graduate education. There are 141 faculty in the Program in Neuroscience; the 74 faculty who are currently most actively involved in graduate education are training mentors on this proposal. Considerable effort has gone into making this program a highly interactive group with extensive formal and informal contacts between students and faculty. Graduates of this program go on to distinguished careers in biomedical research and make substantial contributions to a growing understanding of neuroscience.

Summary The nervous system is composed of diverse cell types that function together in circuits to sense the environment and modulate animal behavior. Sophisticated mechanisms exist throughout development to create and differentiate neural cells, to build complex networks of connections between these cells, and to incorporate new cells into these growing networks. Defects in these basic developmental processes can cause neuro-psychiatric diseases including autism, schizophrenia, and seizure disorders. Exciting new approaches such as brain organoid cultures, single-cell sequencing, and enhanced epigenetic approaches (ChIP-Seq and ATAC-Seq) have led to rapid advances in our understanding of neurodevelopmental processes and diseases. The 2018 Gordon Research Conference (GRC) on Neural Development in Newport, Rhode Island will bring together an international group of scientists that have made breakthroughs in our understanding of nervous system development. Topics to be covered at the meeting include neurogenesis and fate specification, genetic and epigenetic impact on neural development, establishing and maintaining neuronal networks, stem cells, glial cells, and computing neurodevelopment. The meeting is also designed to highlight recent technical advances that have rapidly propelled the field forward, including a diversity of experimental approaches and model systems, from C. elegans to mammals. The Neural Development GRC has been held biennially since 1981 and has become the premier meeting in the field for trainees, young investigators, and senior colleagues. As with many GRCs, it is centered around talks and poster sessions in the mornings and evenings, with extensive time for social and scientific interactions in the afternoons. Our initial slate of speakers is an excellent mix of very promising young (11) and mid-career scientists (3), along with outstanding senior researchers (16). In addition several trainees and young investigators will be invited to give short presentations based on their submitted poster abstracts. Additional features unique to this meeting are lunchtime discussions with trainees on career-relevant issues, and a Power Hour (sponsored by the GRC) to promote the advancement of women in science. The main conference will be preceded by a Gordon Research Seminar (GRS), a two-day event that is organized and run by trainees. The GRS will feature a single keynote address by Chris Doe (University of Oregon); all other talks and posters will be presented by trainees. The GRS, introduced four years ago, is very popular among trainees. We anticipate that this meeting will foster extensive interactions and collaborations between scientists at all stages of their careers, expose attendees to exciting new breakthroughs in the field, and allow us to frame the next exciting set of questions to advance our understanding of nervous system assembly.