Lloyd Greene - US grants

The overall objective of this project is understand the mechanisms whereby trophic factors such as NGF promote the differentiation, maintenance, plasticity, survival and repair of neurons. Past studies indicate that transcriptional regulation plays an important role in the NGF mechanism. However, our understanding of the identities and roles of the genes regulated by NGF is very incomplete. We hypothesize that 1) there are many known and unknown genes that are transcriptionally regulated by NGF that have yet to be recognized as such, 2) that such genes will be detected by the comprehensive SAGE analyses proposed here, 3) that such genes play major roles in the mechanism of NGF actions and 4) that such roles can be determined by deduction of function from sequence analysis combined with experimental manipulation of gene expression. The specific set of studies proposed here will thus seek to comprehensively detect and identify genes regulated by NGF and to test their functional roles in NGF actions. Four aims are given: (1) To carry out comprehensive analysis of NGF-regulated gene expression in PC12 cells by high-throughput SAGE analysis. (2) To Extend PC12 cell SAGE findings to neurons, i.e., to determine whether genes regulated by NGF in PC12 cells are also regulated in cultured neonatal sympathetic neurons and cultured adult sensory neurons as well as in neonatal sympathetic neurons in vivo. (3) To carry out functional analysis of NGF-regulated genes by evaluating multiple classes of NGF responses in cultured PC12 cells and neurons in which expression/activity of NGF regulated gene products is either enhanced or inhibited. (4) To Develop and disseminate (via the Web) comprehensive SAGE data bases of basally-expressed and NGF-regulated genes in PC12 cells and other neuronal systems. Understanding how neurotrophic factors like NGF work will provide insight into a wide variety of key brain functions and has the potential to lead to design of pharmaceutical agents for the treatment and prevention of a number of maladies affecting the nervous system

DESCRIPTION (provided by applicant): The over-arching aim of this project is to uncover shared mechanisms that govern developmental and pathologic neuron death and to apply this knowledge to formulate treatments for stroke, trauma and neurodegenerative disorders. The proposed studies will focus on the apoptotic cell cycle pathway studied in depth under this grant that plays a required role in a variety of developmental and pathological neuron death paradigms. In this pathway, the cyclin-dependent kinase-4 (Cdk4) is activated in neurons in response to apoptotic stimuli. This is turn phosphorylates Rb pocket protein family members causing them to dissociate from gene-repressor complexes with the E2F transcription factors. Dissociation of Rb-E2F repressor complexes leads to derepression and induction of Myb transcription factors that bind to and activate the promoter for apoptotic proteins including Bim. Induction of Bim promotes release of mitochondrial proteins and activation of caspases, resulting in neuron death. This proposal focuses on the mechanism by which apoptotic stimuli activate Cdk4 and in particular on the cdc25a phosphatase. During the cell cycle, cdc25a activates Cdk4 by removing an inhibitory phosphate group. We will assess the hypothesis that cdc25a activity is elevated by apoptotic stimuli in neurons and that this is an apical event in triggering the apoptotic cell cycle pathway. Preliminary supportive data include findings that cdc25a protein and transcripts are elevated in neuronal cells by several different apoptotic stimuli, and that knockdown or chemical inhibition of cdc25a is neuroprotective. There are two specific aims. 1). To test, in in vitro models, the hypothesis that cdc25a plays a proximal role in developmental and pathological neuron death and to dissect the up- and downstream elements of the apoptotic cell cycle cascade in which it partakes. This will involve a variety of cell cultue systems and models for developmental (NGF deprivation) and pathological (AD, PD) neuron death. We will explore a) the required role of cdc25a in neuron death in our various models, b) the extent of cdc25a regulation by apoptotic stimuli, c) the death cascade components that lie downstream of cdc25a, and d) the upstream mechanisms by which cdc25a expression and activity are regulated and how these are engaged by apoptotic stimuli. We will be particularly keen to identify feed-forward death loops involving cdc25a. 2). To test the hypothesis that cdc25a is required for developmental neuron death in vivo. Using the paradigm of developmental neuron death in superior cervical ganglia, we will query whether a) cdc25a levels are induced in dying neurons, b) selected downstream cell cycle pathway components are co-elevated in such neurons, c) biologically-tolerated chemical cdc25a inhibitors suppress neuron death in this system and d) whether such death is inhibited by selective genetic ablation of cdc25a in sympathetic ganglia. Current findings indicate that cdc25a is a druggable target whose in vivo inhibition has little side effects. Thus success of the current aims has a clear potential to lead to development of therapies for pathologic neuron death.

The long-term goal of the studies proposed here is to identify genes that play roles in the degeneration and death of dopaminergic neurons. Such genes and their products have the potential to serve as targets for therapeutic intervention. To accomplish this objective, a powerful technique designated SAGE (Serial Analysis of Gene Expression), will be used for comprehensive analysis of gene regulation in a convenient cell culture model of monoaminergic neuronal death, the 6-OHDA-treated rat PC12 cell. This approach will be used to compare the genes expressed in neuronally differentiated PC12 cells before and at several times after exposure to 6-OHDA. Analysis of the SAGE data will indicate the degree to which specific genes are regulated and will provide sequence information that can be used for their identification and/or cloning. A following set of studies will determine whether such regulated genes are present in dopaminergic neurons in the normal rat brain and whether their expression is regulated during death. This will be achieved by using in situ localization and immunocytochemistry to detect the transcripts and/or their products in neurons within the substantia nigra of normal rats and in rats in which death of such neurons occur during normal development or is evoked by exposure to 6-OHDA or by target injury. Regulated genes detected in the SAGE PC12 cell screen and also in rat brain substantia nigra neurons will then be examined for presence and regulation in Parkinson's Disease. This will be carried out by immunocytochemical localization using material available through the Neuropathology Core of our Center for Research on Alzheimer's Disease and other Neurodegenerative Disorders. Material to be examined will include sections from PD brains, age-matched controls without neurodegenerative disease and brains from patients with other neurodegenerative disorders. A fourth set of studies will exploit cell cultures of PC12 cells and sympathetic neurons to examine the roles of the regulated genes in death evoked by 6-OHDA and other means. This will be accomplished by a variety of strategies including over-expression, down-regulation and interference with function. Because it is anticipated that a large number of regulated transcripts may be detected, a number of criteria will be used to prioritize those genes that will be studied at each step of the project.

This project's overall goal is to identify genes that undergo abnormal levels of expression in neurons affected by Parkinson Disease (PD) and that contribute to their death. The rationale is that neuron death in models of PD and other neurodegenerations requires transcription-dependent gene regulation. Once such genes are recognized and their causal roles in neuron death established, they will become potential targets for preventive/ameliorative therapy in PD. During the last period, we used SAGE to detect approximately 1,200 transcripts (out of 14,000) that are significantly up-regulated in a cell culture PD model. Among other findings, the SAGE profile revealed that PD mimetics induce an endoplasmic reticulum stress response that may contribute to death. For the next period, we propose the following specific aims: 1 To continue to "mine" our SAGE data of 6-OHDA responsive genes. Bioinformatics will be used to match additional SAGE tags to known transcripts and to provide updated assessments of the potential functional roles of the regulated genes in neuronal death and/or PD. 2. To continue studies of the functional roles of select 6-OHDA-responsive genes identified by SAGE. Gene selection will reflect deduced potential relevance to PD and neuron death. Initial emphasis will be on ER stress genes and in particular on the pro-apoptotic transcription factor CHOP. Eight additional regulated genes have been chosen for initial emphasis. Studies will include loss- and gain-of-function experiments with PC12 cells and sympathetic neurons cultured from wild-type and mutant mice. When suitable animal models (null or transgenic) become available, these will be exploited with our collaborators Drs. Burke and Przedborski. 3. To continue examining whether responsive genes detected in our SAGE study are also up-regulated in animals models of PD and in PD tissue. Animal models will be examined collaboratively with Drs. Burke and Przedborski. PD and control tissue will be examined by a) immmunohistochemistry of SNpc and sympathetic neurons (SCG) and b) by quantitative PCR using RNA from such neurons harvested by laser capture microdissection. 4. To use data generated by SAGE or MPSS of control and PD neurons to detect additional regulated transcripts for studies of causal roles in neuronal death in PD. This longer-range aim that will seek to exploit gene profiling of control and PD neurons. Comparison of profiles from SCG, SNpc and PC12 cells will provide a powerful filter for identification of genes for functional in vitro and in vivo analyses as described above.

This project's overall goal is to identify genes that undergo abnormal levels of expression in neurons affected by Parkinson Disease (PD) and that contribute to their death. The rationale is that neuron death in models of PD and other neurodegenerations requires transcription-dependent gene regulation. Once such genes are recognized and their causal roles in neuron death established, they will become potential targets for preventive/ameliorative therapy in PD. During the last period, we used SAGE to detect approximately 1,200 transcripts (out of 14,000) that are significantly up-regulated in a cell culture PD model. Among other findings, the SAGE profile revealed that PD mimetics induce an endoplasmic reticulum stress response that may contribute to death. For the next period, we propose the following specific aims: 1 To continue to "mine" our SAGE data of 6-OHDA responsive genes. Bioinformatics will be used to match additional SAGE tags to known transcripts and to provide updated assessments of the potential functional roles of the regulated genes in neuronal death and/or PD. 2. To continue studies of the functional roles of select 6-OHDA-responsive genes identified by SAGE. Gene selection will reflect deduced potential relevance to PD and neuron death. Initial emphasis will be on ER stress genes and in particular on the pro-apoptotic transcription factor CHOP. Eight additional regulated genes have been chosen for initial emphasis. Studies will include loss- and gain-of-function experiments with PC12 cells and sympathetic neurons cultured from wild-type and mutant mice. When suitable animal models (null or transgenic) become available, these will be exploited with our collaborators Drs. Burke and Przedborski. 3. To continue examining whether responsive genes detected in our SAGE study are also up-regulated in animals models of PD and in PD tissue. Animal models will be examined collaboratively with Drs. Burke and Przedborski. PD and control tissue will be examined by a) immmunohistochemistry of SNpc and sympathetic neurons (SCG) and b) by quantitative PCR using RNA from such neurons harvested by laser capture microdissection. 4. To use data generated by SAGE or MPSS of control and PD neurons to detect additional regulated transcripts for studies of causal roles in neuronal death in PD. This longer-range aim that will seek to exploit gene profiling of control and PD neurons. Comparison of profiles from SCG, SNpc and PC12 cells will provide a powerful filter for identification of genes for functional in vitro and in vivo analyses as described above.

Seeinstructions): This project is based on the rationales that neuron degeneratiDn and death, irrespective of the initiating causes, require transcriptic nal induction of death-associated genes and that such genes and the pathways that are up- and down-stre>am of them are therefore potential ta rgets for therapeutic intervention in Parkinson's Disease (PD). There are four interlinked steps in our prograrn. The first was a '"discovery" step that used "SAGE: analysis to identify pro-apoptotic genes induced in a cellular model of PD. Among the induced genes was one designated RTP801and this was found to be both sufficient and necessary for death in our cellular PD models. The set;ond step was "verification" of relevanee to PD and demonstrated that RTP801 is elevated in dopaminergic neurons of PD patients. The third step involved studying the "mechanisms" by which RTP801 is regulated and by which it mediates neijron degeneration and death. This revealed that RTP801 acts in part b;/ suppressing activation of the cellular kinase mTor and that it promotes neuron death by leading to inactivation of the neuronal survival-prc moting kinase AKT. Moreover, mechanism studies with the mTor inhibitor rapamycin show that mTor also acts upstream of RTP801 and is required for its induction, both in cuIture and in an animal model. The Ialter findings led us to preliminary incursions into the fourth step of the program, "therapeutics". Raparnycin, which suppresses RTP801 induction, protects neuronal cells from PDmimetics inculture and inanainimal model. In aggregate, these findings advanced our long-term goal of developing a therapeutic strategy for suppressing neuron degeneration and death in PD and form the basis for the following spejcific aims: Aim 1: To define the pathways that govern RTP801 indueDtion and by which it triggers degenei[unreadable]ation and death a. To test in PD the "AKT hypothesis" that elevated Fn"P801 promotes neuron degeneration and death by depleting activated AKT b. To identify the apoptotic signaling pathways that are activated downstream of RTP801 induction and AKT depletion and that mediate neuron death in PD models and in PD c. To identify the upstream transcription factors that induce RTF3801 in PD models and in PD. Aim 2: To further explore (in culture and animal models) the role of mTor signaling in RTP801 induction and to further evaluate the potential of mTor inhibition as a therapeutic for PD. Aim 3: To examine the role of RTP801 in neuron degeneration and death in a genetic model of PD. Aim 4: To test the hypothesis that the gene Trb3 that is induced in our cellular model of PD contributes to neuron death by acting along with RTP801 to s uppress AKT activation. RELEVANCE (Seeinstructions): Parkinson's disease is a progressive and debilitating neurodegenerative disorder. The ultimate goal of these studies is to identify therapeutic targets for treatment of PD and to point the way o drugs that can be developed towards this end. Our program has thus far found several potential targets in this regard as well as a drug for potential therapeutic development. The proposed work will continue to exploit these findings.

DESCRIPTION (provided by applicant): Support is requested for a training program to support the interdisciplinary research training and professional career development of advanced graduate students in their third, fourth and fifth years of an existing University-wide, degree-granting Doctoral Program in Neurobiology and Behavior at Columbia University. Neuroscience is one of the most rapidly growing areas of contemporary biomedical research and one in which basic findings are increasingly arising from interdisciplinary and collaborative research and are being translated into clinical advances. The program arises from the recognition that there are training opportunities that are of special relevance and importance to advanced graduate students in neuroscience and that there is a changing landscape for preparing for, finding and sustaining careers in biomedical and neuroscience research. In this context, the themes of the program will be to encourage and support our students in carrying out novel, interdisciplinary and collaborative thesis research projects not easily supported by existing programs and to provide them with professional career development training. Research training will be provided by the 88 faculty in the program, including leaders in various areas of neuroscience with a broad span of research interests. To further facilitate interdisciplinary training, students will be supported for short visits to laboratories outside of the New York area for the purpose of learning and applying novel techniques not available locally. Enhanced professional career development training will include a newly instituted course in this area for advanced graduate students in our program, the augmentation of a currently existing course in translational neuroscience, and creation of opportunities for students to receive guidance on careers in translational neuroscience from appropriate physician/scientists within our institution. Total student enrollment in the overall Neurobiology and Behavioral Program is approximately 80. Support is requested for 4 advanced students initially, with a yearly increment of 1 student to reach a total of 8 by year 5. Students will be eligible for the program after the first two years of intensive instructional experience. Appointment will be competitive and selection will be by a designated faculty committee. RELEVANCE: The goal of the proposed program is to support novel, interdisciplinary and collaborative research as well as professional career development for advanced students in neuroscience. Neuroscience is one of the most rapidly growing areas of contemporary biomedical research and one in which basic findings are being translated into clinical advances in neurological and psychiatric disorders.

? DESCRIPTION (provided by applicant): We request continued support for a training grant for advanced (3rd-5th year) students in the well-established Columbia University-wide Doctoral Program in Neurobiology and Behavior. Support for five students is requested. The impetus for initiating and continuing this training program is the desire to provide the best possible preparation of our students to fulfill productive careers that draw on their scientific training. I particular, we will support a cadre of students carrying out cross-disciplinary research that has translational significance and relevance to neurological diseases. We will further favor and encourage such research that is directed by co-mentors with different experimental expertise. In addition, we will provide out-of-laboratory training of these and other students in the program via a continuing course in Professional Skills and a course in Biology of Neurologic and Psychiatric Disorders. We will also add several new added value components to the program via an association with the newly inaugurated Columbia Translational Neuroscience Initiative (CTNI). The CTNI is dedicated to serving as an umbrella organization to coordinate and enhance basic and translational neuroscience research at Columbia. Aside from the benefits conferred by its overall mission, the CTNI will specifically organize and run a mentored data presentation forum that our supported students will participate in along with other doctoral and post-doctoral trainees. It will also offer our students presentations by clinical faculty of patiens with neurological disorders. These training opportunities are designed to be leveraged so that they benefit our supported trainees as well as other neuroscience-oriented students at Columbia. The program initially will be directed by the current PD Lloyd Greene, and a co-PD, Gil Di Paolo. Dr. Greene will phase out after the first year of the renewal period and Dr. Di Paolo will become sole PD. This arrangement is designed to permit a smooth transition of the program leadership. The program will have 52 mentors at both junior and senior levels who work in area of relevance to translational neuroscience. Oversight will be provided by the directors of the overall Neurobiology and Behavior program, by an outside committee and by feedback from current and past students supported by the program. In the first 4 years of the program, 11 students have been supported with mentorship/co-mentorship by 13 different faculties. Publication records, thesis completion, retention rates, success in gaining outside support, continuation in science careers, increasing numbers of qualified and co-mentored applicants, and student feedback all indicate that the program is meeting its goals. This and additional considerations support the request to increase the number of supported students to five from the current number of four.

The ultimate goal of the proposed research is to create innovative means to halt the progression of neuron degeneration in Parkinson's disease (PD). Present treatments alleviate the symptoms of PD, but not its advancement and there is therefore a major need for new ideas and approaches to slow or stop the relentless degeneration of neurons that occurs in this disorder. The core idea here is that irrespective of the initiating causes of PD, neuron degeneration and death are promoted by dysregulation of shared distal pro-death pathways and that targeting these pathways and/or their components has the potential to stop or slow disease progression. This renewal continues to focus on the protein Trib3. Our findings have shown that Trib3 is induced by multiple cellular stresses associated with PD and is elevated in dopaminergic substantia nigral neurons of PD patients as well as in multiple cellular models of PD. Trib3 is both sufficient and necessary to promote neuron degeneration and death in cellular models of PD and its deletion or suppression in such models provides protection from neurite degeneration and death. Examination of the upstream regulatory pathway of Trib3 induction in PD models reveals necessary involvement of the transcription factors ATF4 and CHOP/DDIT3 and preliminary data show that interference with this pathway by the drug ?adaptaquin? blocks Trib3 induction and neuron death and degeneration in cellular PD models. Regarding mechanism, our studies show that Trib3 interacts with Parkin (a neuroprotective protein whose loss or dysfunction leads to an early onset form of PD in humans and to neuron degeneration in cell and animal models) in living cells and that this association leads to Parkin depletion via the COP1 E3 ligase. These findings lead to the hypothesis that the chronic cellular stresses that initiate the path towards neuron degeneration and death in PD elevate Trib3 expression and that this contributes to the slow and inexorable progression of the disease at least in part by diminishing levels of Parkin. To exploit these observations so as to move forward towards targeting Trib3 for the purpose of alleviating the progression of PD in patients, 3 specific aims are proposed. These are: 1) To test the hypothesis that conditional deletion of Trib3 in substantia nigral dopaminergic neurons of living mice will provide protection in models of Parkinson's disease. PD models will include treatment with 6-OHDA and a- synuclein over-expression/exposure to a-synuclein fibrils. 2) To test the hypothesis that Trib3 contributes to neuron death in PD by interacting with and depleting neuronal levels of the protective protein Parkin and to define the bases for this interaction so as to provide strategies to disrupt this association for potential therapeutic purposes. 3) To test the hypothesis that interference with Trib3 induction by the drug ?adaptaquin? will protect neurons in cellular and animal models of PD. The successful outcome of these studies will promote development of a Trib3-targeted therapy that alters the course of degenerative progression in PD.