Wilma J. Friedman - US grants

DESCRIPTION: (Applicant's Abstract): Inflammatory cytokines are present in the brain during development and consequent to a variety of neurological insults ranging from local lesions to neurodegenerative disease. The functions subserved by cytokines in the nervous system are poorly understood, however these molecules may mediate the effects of inflammatory responses on neural function, and provide a critical link between the immune and nervous systems. Initial studies from our lab and others have shown that interleukin-1 (Il-1), an important immunomodulatory cytokine, regulates expression of nerve growth factor (NGF), a neurotrophic factor critical for neuronal survival and function. The overall objective of this proposal is to define mechanisms governing the interaction between cytokines and neurotrophic factors in the brain. To identify potential cytokine-responsive cellular populations, expression of receptors for Il-1 and the related cytokine TNF will be localized in the adult and neonatal brain using in situ hybridization. To determine whether neurons and/or glial cells respond to cytokines, hippocampal cultures will be grown as separate neuronal or glial cultures and exposed to Il-1 and related inflammatory cytokines TNF and Il-6. NGF mRNA, expressed by both hippocampal neurons and astrocytes in culture, will be examined in the separate populations for responsiveness to cytokines. In addition, other members of the neurotrophin gene family, BDNF and NT-3, which are also expressed in the hippocampus, will be examined for regulation by inflammatory cytokines. Mechanisms underlying regulation of NGF by Il-1 will be characterized. The Il-1 signal transduction pathway will be examined by assessing the involvement of different second messenger systems, and by investigating the role of transcription factors such as AP-1 and NFkappaB. Thus, we propose to further define the mechanisms by which immunomodulatory cytokines influence neuronal function.

DESCRIPTION (provided by the applicant): Neurotrophins have long been known to support the survival and differentiation of many neuronal populations through interactions with the trk tyrosine kinase receptors. Recent studies from our laboratory and others have demonstrated that neurotrophins can also elicit cell death via the p75 receptor. Thus, whether a neuron survives or dies in response to neurotrophins may depend on a balance of signaling pathways activated by these different receptors. Although p75 is transiently expressed in many CNS neuronal populations during development, this receptor is not widely expressed in the normal adult brain. However, after damage in the brain expression of p75 can be detected in numerous neurons, suggesting that upregulation of this receptor may be part of an injury or stress response. Since production of NGF is also induced in the brain by many types of damage, we hypothesize that NGF interacts with p75 to eliminate neurons that are compromised by damage. In this grant application we propose to investigate whether the p75 receptor is expressed on dying neurons in vivo as a consequence of pilocarpine-induced seizures. We will compare the number of dying neurons after seizure in normal mice with p75-/- mice to assess the role of this receptor in mediating neuronal loss. These in vivo studies will be accompanied by studies on hippocampal neurons in vitro to investigate the mechanisms of p75-mediated neuronal death. Thus, the goal of these studies is to define the role of the p75 neurotrophin receptor in mediating cell death in hippocampal neurons, particularly under conditions of brain injury.

DESCRIPTION (provided by applicant): Multi-year funding is requested to partially support a new Gordon Research Conference entitled "Glial Biology: Functional Interactions Among Glia & Neurons". The first of these biennial conferences is Feb. 23-28, 2003 in Ventura CA. It will focus on complex issues at the frontier of glial biology involving neuron and glib interactions, with strong emphasis on cell-cell communication. Major conference themes include glial control of neuronal functions, neuronal control of glial activities, formation and functions of glia-neuron and glia-glia networks, neuron-glia & glia-glia interactions related to neurotransmission, trophic interactions and disease processes, and modeling of spatio-temporal processes. There are three primary conference objectives. (1) Initiation of a continuing interactive dialogue between scientists in different fields of glial biology, glia-neuron, and glia-glia interactions by providing a long-term forum for presentation and discussion of cutting-edge work in selected fields to a diverse group of leading researchers. (2) Suggest new integrative approaches to solve timely problems in brain function and disease, identify controversial issues that need new approaches to be resolved and foster collaborative projects in multi-disciplinary research. (3) Bring to the attention of the neuroscience community the importance of the glial cells and their interactions with other cell types in normal brain functions and disease and in modeling interactions of these cell types in functional systems of neuronal activity. The proposed program (9 oral & 4 poster sessions) has 27 invited speakers from the international community within the sub-disciplines of glial biology. To ensure vigorous discussion participants are limited to 135, with representation of senior and junior investigators, gender, minorities, and geographic area. The conference site has disability access. Federal funds are essential to support conference/travel costs for 42 invited participants (including awards for 14 meritorious junior researchers): NIH funds are requested to support 16 participants in each of 3 biannual conferences. "Glia & other non neural cells" is one of seven priority cross-cutting topics central to the NINDS strategic research questions for neuroscience in the new millennium. This Glial Biology Conference provides an international forum for emerging, innovative research related to glia functions and cell-cell interactions in health and disease.

DESCRIPTION (provided by applicant): The overall aim of this proposal is to dissect the physiological relevance of the different pathways activated by p75ntr and to elucidate novel functions using a genetic approach combining in vitro and in vivo studies. We hypothesize that p75ntr has the ability to generate diverse, and sometimes contradicting, signals depending on the cell context and the nature of the activating ligand. In order to tackle the complexity of p75ntr function, we propose a structure-function approach to dissect those different signals, and a genetic approach to investigate their biological importance in relevant cell types, both in vitro and in vivo. In order to reveal novel p75ntr functions, we will generate gain-of-function alleles of this receptor and characterize their function in cell lines and primary cultures, and in vivo by knock-in expression in transgenic mice. Currently, only loss-of-function models for investigating in vivo roles of p75ntr are available, the analysis of which may be complicated by redundant and compensatory pathways. A gain-of-function approach may help to reveal novel in vivo functions of this receptor. In order to dissect the physiological relevance of the different pathways activated by p75ntr, we will generate loss-of-function alleles of this receptor capable of activating some pathways but not others, and characterize their function in vitro and in vivo by knock-in expression in transgenic mice. Further mechanistic and functional insights may be obtained by combining distinct sets of gain- and loss-of-function mutations in individual p75ntr molecules. We suggest that these types of studies will help to link distinct signaling pathways and downstream effectors to specific biological activities and phenotypes. Given the overall medical importance of the ligands, signaling pathways and biological activities of p75ntr, a better understanding of the mechanisms and in vivo functions elicited by this receptor is essential, particularly as agonists and antagonists are being considered for therapeutic purposes in nerve damage, stroke and neurodegenerative diseases.

Neurotrophins are a family of factors that influence a multiplicity of functions in the central nervous system that range from supporting neuronal survival to eliciting apoptosis, regulating synaptic function, and influencing axonal growth. These distinct functions are mediated by the interactions of the factors with two distinct receptors, a member of the Trk family of receptor tyrosine kinases and the p75 neurotrophin receptor (NTR). It has recently been shown that neurotrophin precursors, proneurotrophins, selectively bind and activate p75NTR, effectively eliciting apoptosis, while the mature (cleaved) neurotrophins preferentially activate Trk signaling, which supports neuronal survival. Many neurons express Trk receptors and p75NTR, especially after injury when p75NTR is upregulated. In the proposed studies we will investigate how the signaling pathways activated by these different receptors interact to determine how neurons respond when exposed concomitantly to mature and proneurotrophins, as occurs after injury. Our previous studies have shown that p75NTR initiates apoptotic signaling while supressing Trk-mediated survival signaling. Our proposed studies will explore these mechanisms further. One key determinant of whether Trk or p75NTR is activated depends on which form of the ligand, mature or proneurotrophins, are available to bind the different receptors. Since proneurotrophins can be secreted and cleaved extracellularly, we will investigate the regulation of proneurotrophin cleavage, particlarly after seizures when we have detected increases in proneurotrophins in the CNS, and induction of p75NTR-mediated apoptosis. The p75NTR can function as a co-receptor with several other receptors to mediate different functions. The high-affinity binding of proneurotrophins to is due to the interaction with a co-receptor, sortilin, and our preliminary studies have shown that both receptors are required for proneurotrophins to elicit apoptosis. The signals that determine the association of p75NTR with its different co-receptors are unknown, and we will investigate this question in the proposed studies. In sum, these studies will provide greater insight into the multifunctional effects of neurotrophins in the brain, particularly under inflammatory conditions that occur after injury or in disease.

DESCRIPTION (provided by applicant): Many types of insults or injury lead to seizures and neuronal loss, which ultimately can lead to devastating and intractable long-term consequences, including cognitive impairment, chronic epilepsy and disability. Our labs have identified a central role of neurotrophins, particularly proNGF in mediating neuronal apoptosis following severe seizures (status epilepticus;SE). Neurotrophins, initially characterized for their survival and differentiative actions on neurons, are initially synthesized as precursors that are cleaved to release C-terminal mature forms that bind to Trk receptors to promote neuronal survival, and synaptic actions. Recent studies suggest that the precursor form of NGF (proNGF) acts as a distinct ligand by binding to a receptor complex of p75 and sortilin to initiate neuronal death. Our labs have demonstrated that SE rapidly induces p75 and proNGF in the hippocampus, in rodent models. Importantly, infusion of function blocking anti-proNGF antibodies, or genetic deletion of p75, rescues neuronal death following SE, providing a mechanism and target for therapy to prevent neuronal loss after SE, and its consequence, epileptogenesis. Here we extend these collaborative studies to utilize novel knock-in mouse lines that expressed HA-epitope tagged NGF, or proNGF under its endogenous promoter elements. These animals circumvent the current limitations in sensitivity of reagents, and markedly enhance our ability to detect endogenous NGF. Thus we will be able to identify cell specific and temporal patterns of NGF/proNGF synthesis in neurons and glia under basal conditions and after SE. Using the proNGF mouse, we will quantitatively evaluate the effects of augmented proNGF following SE induction, to assess whether excess proNGF leads to exacerbated neuronal cell death. We will also use video-EEG to assess the consequence of excess proNGF on the development of epilepsy. Finally, and most important, we will investigate whether antagonists of proNGF, identified in a high through-put drug screen, can prevent or attenuate neuronal death following SE and prevent the onset of epilepsy. These antagonists consist of FDA-approved drugs that have minimal toxicity and efficiently block proNGF-induced apoptosis in cultured neurons. The possibility of identifying approved drugs that have the capacity to prevent neuronal death from severe seizures has significant potential for clinical application, both for epileptic patient populations, and for those suffering seizures as a consequence of brain trauma or stroke.

DESCRIPTION (provided by applicant): Inflammation occurs as a consequence of brain injury, and results in the production of inflammatory cytokines, which contribute to the progression of the insult. One of the most potent inflammatory cytokines is interleukin-1 (IL-1), which is induced by many types of brain injury and can potentiate neuronal loss. IL-1 inhibitors have been suggested as therapeutic tools following brain injury to attenuate neuronal loss. However, IL-1¿ is also expressed in the brain under physiological conditions, and may play a role in mediating normal hippocampal function. We have previously demonstrated that IL-1¿ activates distinct signaling pathways in neurons versus glial cells in the brain. Therefore, to assess the consequences of inflammation that occur following injury, and the potential therapeutic value of inhibiting inflammation, it is critical to understand the cell-specific consequences and to distinguish physiological versus pathophysiologial roles for this key cytokine. IL-1 signals via a receptor complex composed of the type 1 IL-1 receptor (IL-1R1) and the IL-1 Receptor Accessory Protein (IL-1RAcP). A novel isoform of the IL-1RAcP, termed AcPb, has been identified that is expressed exclusively in CNS neurons, providing a new opportunity to investigate the mechanisms governing neuron-specific IL- 1 actions. To identify the function of the neuron-specific IL-1RAcPb we will establish neuronal cultures from AcPb-/- mice compared to wild type neurons and to neurons lacking both isoforms of the protein (AcP-/-). The effects of IL-1¿ on these neurons will be analyzed to determine the role of AcPb in mediating neuron-specific signaling pathways, and the consequences for neuronal function.

? DESCRIPTION (provided by applicant): This proposal is to provide support for junior investigators, post-doctoral fellows and students to attend the 12th Gordon Research Conference (GRC) on Neurotrophic Factors at Salve Regina University, Newport RI from May 31 - June 5, 2015. The meeting will be attended by approximately 150 participants and will feature work on diverse neurotrophic factors, their functions and mechanisms of action, and their potential therapeutic capacity. Neurotrophic factors are extracellular proteins that act on developing or mature neural cells to promote cell survival, neural differentiation, and circuit connectivity and function. Defects in neurotrophic factor activities are linked to a broad variety f neurological conditions, including neurodegenerative diseases, neural injury, neuropsychiatric disorders such as autism and cognitive dysfunction, and even addictive behaviors. The objectives of this grant are to provide support for a meeting that will: 1. Foster interactions amongst diverse basic and clinical researchers who study neurotrophic factors in health and disease. Neurotrophic factors is a highly multidisciplinary field with researchers ranging from molecular/cell biologists to systems biologists, electrophysiologists, and human biologists interested in brain disorders. The philosophy of this conference is that the very best science will result from collaborations and interactions amongst these diverse researchers. Thus, a primary objective of the upcoming GRC is to foster and strengthen those interactions, leading to scientific excellence and providing a chance for young investigators to learn about the important questions and challenges. 2. Achieve a sense of community amongst researchers studying neurotrophic factors. Science is an international endeavor, and the field of neurotrophic factors is no exception. This meeting will feature international scientists in the field, and will highligh many of the accomplished scientists in the field who are women or members of under-represented minorities. In so doing, this meeting will achieve its second objective, to enhance the sense of a global community, and to break down barriers to the interactions that allow research to move from the bench to the clinic. 3. Provide opportunities for young researchers to present their work and interact with the broad neurotrophic factors community. This meeting will encourage communication of ideas at the frontiers of the area, including a session of hot topics, short talks presented predominantly by students and postdoctoral fellows. Funds from NIH will be used to enable these young investigators to attend the meeting and present their novel findings in an exciting and collaborative setting.

? DESCRIPTION (provided by applicant): Regulation of proliferation, differentiation, and migration of neural progenitor cells is critical for the normal development of the nervous system. Many factors can influence these events, including the neurotrophin family of factors. These processes have been extensively studied in cerebellar granule cell progenitors (GCP), the most abundant cell type in the brain, which undergo much of their development postnatally. GCPs proliferate in external granule layer (EGL), a transient layer of the cerebellum, and migrate internally to form the internal granule layer (IGL). In the external part of the EGL these cells proliferate, while in the internal part of the EGL the cells exit the cell cycle and start to migrae toward the inside of the developing cerebellum. The p75 neurotrophin receptor (p75NTR) is highly expressed in the EGL during development of the cerebellum, and is absent once the cells begin to migrate. The function of p75NTR in this developing neuronal population has not been defined. Our preliminary data demonstrate that this receptor promotes withdrawal from the cell cycle and inhibits migration, and mice lacking this receptor show increased markers of proliferation, delayed cell cycle exit, and an enlarged cerebellum. We propose that p75NTR plays a crucial role in regulating the timing of the transition of granule cell progenitors from a proliferating to a migrating population. The p75NTR-/- mice have structural deficits in the adult cerebellum, and our preliminary data indicate that adult mice lacking p75NTR in the EGL during development show deficits in motor behavior, suggesting that modulation of these developmental events have long-lasting consequences in the adult. Thus, we expect to define novel roles for p75NTR in the developing brain that have long-term consequences for cerebellar structure and function.

ABSTRACT This proposal is to provide support for junior investigators, post-doctoral fellows and students to attend the 13th Gordon Research Conference (GRC) on Neurotrophic Factors at Salve Regina University, Newport RI from June 4-9, 2017. The meeting will be attended by approximately 150 participants and will feature work on diverse neurotrophic factors, their basic mechanisms of action as well as their functions in behavior and disease. Neurotrophic factors are extracellular proteins that act on developing or mature neural cells to influence cell survival, neural differentiation, and circuit connectivity and function. Defects in neurotrophic factor activities are linked to a broad variety of neurological conditions, including neurodegenerative diseases and neural injury, as well as neurodevelopmental and neuropsychiatric disorders such as autism and cognitive dysfunction. The objectives of this grant are to provide support for a meeting that will foster interactions amongst diverse researchers who study neurotrophic factors in health and disease. The field of neurotrophic factor research is highly multidisciplinary, with researchers ranging from molecular/cell biologists to systems biologists, electrophysiologists, and human biologists interested in brain disorders. The philosophy of this conference is that the very best science will result from collaborations and interactions amongst these diverse researchers. Thus, a primary objective of the upcoming GRC is to foster and strengthen those interactions, leading to scientific excellence and providing a chance for young investigators to learn about the important questions and challenges. Additionally, this conference will promote a sense of community among researchers studying neurotrophic factors. Science is an international endeavor, and the field of neurotrophic factors is no exception. 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. In so doing, this meeting will achieve its second objective, to enhance the sense of a global community, and to break down barriers to the interactions that allow research to move from the bench to the clinic. Finally, this meeting will provide opportunities for young researchers to present their work and interact with the broad neurotrophic factors community. This meeting will encourage communication of ideas at the frontiers of the area, including a session of hot topics, short talks presented predominantly by students and postdoctoral fellows.