Mohanish Deshmukh - US grants

DESCRIPTION (provided by applicant): Programmed cell death (PCD), which results in apoptosis, occurs widely during neuronal development and is also observed in pathological situations of stroke, spinal cord injury, and neurodegenerative disease. The mechanism of neuronal PCD has been extensively studied in sympathetic neurons that undergo apoptosis after nerve growth factor (NGF) removal in culture. A critical factor regulating apoptosis in many cells is the cytochrome c-dependent activation of caspases. Although necessary in sympathetic neurons, cytochrome c release is not sufficient to induce apoptosis after NGF deprivation. We have recently demonstrated that a novel, uncharacterized event, called the "development of competence," is needed, along with cytosolic cytochrome c to induce caspase activation and apoptosis in these neurons. We shall examine whether the development of competence event is also important in other models of neuronal apoptosis and test the specific hypothesis that the requirement of development of competence to induce apoptosis is a phenomenon unique to postmitotic cells. We shall also examine the signaling pathway activated after NGF deprivation that leads to the development of competence. Since our preliminary results suggest that the c-jun-N-terminal kinase (JNK) signaling pathway is important in regulating competence in sympathetic neurons, we shall focus specifically on components of this signaling pathway. Lastly, we shall examine the molecular mechanism of development of competence. Our recent data suggest that competence may be controlled by an inhibitor of apoptosis protein (IAP) like activity. We shall examine this hypothesis and test the specific importance of Smac, a recently identified inhibitor of lAPs, in regulating the development of competence in neurons. These studies will provide an understanding of the biological importance and mechanism of development of competence in promoting neuronal apoptosis. Knowledge of this pathway may also identify targets for the development of strategies to suppress apoptosis and ameliorate the consequences of neuronal injury and neurodegenerative disease.

DESCRIPTION (provided by applicant): The existence of the apoptotic pathway can be both vital and devastating for organisms. While regulated cell death by apoptosis is essential during development and for maintaining homeostasis in organisms, dysregulation of apoptosis is associated with numerous pathological conditions including cancer, neurodegeneration and cardiovascular diseases. Therefore, a cell's ability to suppress the apoptotic pathway, activating it only when necessary, is indeed a delicate balance. Our hypothesis is that there are fundamental differences in how the apoptotic pathway is regulated in mitotic versus postmitotic cells. We propose that in postmitotic cells such as neurons, cardiomyocytes, and myotubes, which have limited regenerative potential and last for the lifetime of organisms, apoptosis is regulated more strictly than in mitotic cells. We have focused our studies on the regulation of cytochrome c- dependent caspase activation because this event is a crucial point of no return for cell death in mammalian cells. We find that whereas cytosolic cytochrome c alone is sufficient to induce apoptosis in many mitotic cells, it is not capable of doing so in the postmitotic neurons, cardiomyocytes and myotubes because of strict control of caspase activation by XIAP. We have also identified another unexpected checkpoint where we find levels of endogenous cytochrome c to be limiting for apoptosis in postmitotic but not mitotic cells. In Specific Aim 1, we will investigate how endogenous XIAP is able to selectively regulate apoptosis in postmitotic but not mitotic cells. We will also examine how XIAP's strict inhibition of caspases is relieved to permit apoptosis in postmitotic cells. In Specific Aim 2, we will examine the mechanism by which endogenous levels of cytochrome c become rate-limiting for apoptosis in postmitotic but not mitotic cells. We will also test the hypothesis that apoptotic signals in postmitotic cells elevate endogenous cytochrome c levels above the threshold necessary for activating caspases. In Specific Aim 3, we focus our attention on senescent cells. We will examine whether the development of senescence engages changes in the regulation of cytochrome c-dependent apoptosis as seen in the terminally-differentiated cells. Understanding how apoptosis is differentially regulated in mitotic and postmitotic cells is clinically significant because it identifies drug targets that could inhibit or activate apoptosis in selective cell types.

Although studies in cell lines have identified the main components of the mammalian apoptotic pathway, the differences in the regulation of apoptosis among various primary cells remain largely unexplored. For example, biochemical and molecular studies have identified the caspase proteases as central executionersof apoptosis and the release of mitochondrial holocytochrome c (heme-attached cytochrome c) as a critical trigger that activates caspases during apoptosis. However, despite the central importance of cytochrome c- mediated caspase activation in mammalian apoptosis, our understanding of how this pathway is regulated in primary cells is verylimited. Our recent results point to a potentially fundamentaldifference in the regulation of caspase activation in primary mitotic versus postmitotic cells. We find that whereas holocytochrome c alone is necessary and sufficient to activate caspases and induce apoptosis in mitotic cells, it is necessary but not sufficient to induce apoptosis in neurons, cardiomyocytes, and myotubes. This difference is striking and is indicative of novel, differential mechanisms of apoptosis regulation in mitotic versus postmitotic cells. Unfortunately however, our understanding of the molecular basis for this difference is limited because the only current method for activating apoptosis at the point of cytochrome c is via single cell microinjection of holocytochrome c. Thus, these experiments are restricted to only those cells that can be microinjected and they are not compatible with large scale biochemical analysis. To overcome these limitations and to develop novel probes for studying apoptosis regulation, we will adapt a cell-free caspase activation assay for high throughput compatibility to screen for small-molecule compounds that trigger apoptosis after the point of mitochondria] cytochrome c release in cells. The development of these chemical probes is significant in two important aspects: a) They are essential tools for discovering the molecular mechanism of the differential regulation of apoptosis in mitotic and postmitotic cells, and b) These probes are predicted to have therapeutic value as reagents that induce apoptosis only in selective cells.

DESCRIPTION (provided by applicant): Regulated neuronal death by apoptosis is a key part of normal brain development. Unfortunately, neuronal apoptosis is also triggered after brain injury or in neurodegenerative disease, where it contributes to the loss of neurons and the resulting neurological deficits. Thus, the neuronal apoptosis pathway is of physiological and pathological importance. Recent studies have reported an age-dependent decline in the incidence of apoptosis in the brain. However, the mechanisms of these changes are not well studied, and its implications on how apoptosis is activated in neuropathological situations in young versus adult neurons are unclear. Our long-term objectives are to understand the molecular mechanisms by which apoptosis is regulated in neurons, with a specific interest in identifying age-related changes in this pathway. Our focus is on the mechanism of activation of caspase proteases, which are the central executioners of apoptosis. A major pathway of caspase activation involves the mitochondrial release of cytochrome c and subsequent formation of the Apaf-1-dependent apoptosome complex. Our results have identified distinct age-dependent changes in which the cytochrome c-dependent caspase activation pathway becomes increasingly inhibited in neurons. For example, we find that caspase activation in neurons during early development is strictly and selectively regulated by XIAP. In Specific Aim 1, we will examine how XIAP's strict control of caspases is overcome to permit caspase activation in physiological (NGF deprivation) and pathological (ER stress) models of sympathetic neuronal apoptosis. We will also examine whether XIAP-deficient neurons are more vulnerable to neuronal injury in vitro and in vivo. We will examine this in neurodegeneration models of mutant superoxide dismutase 1 (SOD1) overexpression and neonatal hypoxia-ischemia. At later stages of development, we find that neurons engage additional mechanisms to inhibit apoptosis. In Specific Aim 2, we will focus on identifying the posttranslational mechanism that inactivates Apaf-1 in neurons during the late period of cerebellar development. Finally, as neurons mature, they appear to turn off the expression of Apaf-1 and caspase-3 altogether. In Specific Aim 3 we will test whether the cell cycle- related protein E2F1, which is known to be expressed in dying neurons, is important for restoring Apaf-1 expression and permitting cytochrome c-mediated apoptosis in mature neurons. Understanding the mechanism by which apoptosis is regulated in neurons is clinically important for identifying appropriate drug targets that would effectively prevent neuronal death after injury or disease.

Small-molecule chemical probes are widely used as research tools to study biological function and disease mechanisms (Strausberg and Schreiber, 2003). Indeed, a major new initiative of the NIH Roadmap project has been the establishment of Molecular Libraries Screening Center Networks (MLSCN) (http://mli.nih.gov/mlscn/index.php). The goals of these centers are to provide academic researchers access to small molecule compound libraries that can be screened using high throughput robotics. Whole-genome RNAi-based libaries are also being used to identify genes involved in a variety of cellular pathways (Ashrafi et al., 2003; Friedman and Perrimon, 2006). However, a major limitation for most researchers is that their biological assays are not compatible or optimized for HTS. This has restricted access for many investigators as the screening centers accept only those assays that are already miniaturized for a multi-well format and shown to produce a robust and reproducible readout that can be quantified with HTS devices. Some considerations important for the development and validation of HTS assays (http://mli.nih.gov/mlscn/index.php) are that the assays should: 1) Be easy to automate with steps such as centrifugation, filtration and extraction avoided; 2) Have demonstrated capability of working reproducibly in a 96-, 384-, or 1536-well plate format; 3) Have signal of sufficient intensity with a signal-to-background ratio of at least 5 and a coefficient of variation (CV) below 10%; and 4) Have a Z'-factor value in the range of 0.5-1.0. These statistical values take into account both the assay signal dynamic range and the data variation (Zhang et al., 1999). The purpose of Core 6 is to facilitate the development of such assays for NINDS qualifying investigators such that the assays are validated for HTS.

DESCRIPTION (provided by applicant): The ability to strictly regulate apoptosis is particularly important for postmitotic cells such as neurons, cardiomyocytes and myotubes because these cells have limited regenerative potential and are maintained for the lifetime of the organism. Failure to restrict apoptosis can result in increased vulnerability to cell death, as seen in many degenerative diseases. Therefore, an understanding of survival mechanisms in postmitotic cells is of physiological and pathological importance. The mitochondrial release of cytochrome c (cyt c) is a crucial event that triggers caspase activation during apoptosis. Here, we have identified a novel mechanism for regulating cyt c-mediated apoptosis engaged by neurons and other postmitotic cells, where cytosolic cyt c itself is targeted for proteasomal degradation. Importantly, while we do not observe cyt c degradation in primary mitotic cells such as fibroblasts, we find mitochondrial-released cyt c to be also degraded in certain cancer cells. As evasion of apoptosis is an important feature of both postmitotic and cancer cells, targeting cytosolic cyt c for degradation could be a shared mechanism used by these cells for survival. In this proposal, our goals are to identify the molecular mechanisms by which cytosolic cyt c is targeted for degradation. In Aim 1, we will define the factors which determine whether or not a cell will target cytosolic cyt c for degradation, including examining whether low levels of Apaf-1 in cells is a key determinant for cyt c degradation. In Aim 2, we will examine cyt c ubiquitination and identify the specific residues of cyt c that are targeted for ubiquitination. Our hypothesis is that ubiquitinated cyt c is not able to bind Apaf-1. We will test this, and examine whether conditions that block cyt c degradation increase the vulnerability of postmitotic cells to apoptosis. Our focus in Aim 3 is to identify the E3 ubiquitin ligase that targets cyt c for degradation. Our preliminary results suggest that cyt c binds to Hsp70 and the E3 Ligase CHIP (Carboxyl Heat shock protein 70-Interacting Protein) in the cytosol. Therefore, we will test whether the ubiquitination and degradation of cytosolic cyt c is mediated by CHIP in postmitotic and cancer cells. PUBLIC HEALTH RELEVANCE: Our plans here are to investigate how the cell death pathway is regulated in mammalian cells. We have discovered a novel mechanism engaged by postmitotic cells such as neurons, cardiomyocytes and myotubes that highly restricts cell death and likely ensures their long term survival. Understanding the survival mechanisms used by postmitotic cells has enormous clinical significance because increased death of these cells is central to the pathology of many neurodegenerative diseases, cardiac pathologies and muscular dystrophies.

DESCRIPTION (provided by applicant): Human embryonic stem (hES) cells have received considerable attention with regards to their regenerative capacity. However, basic biological pathways including apoptosis in hES cells remain largely unexplored. We have started to investigate the apoptotic pathway in hES cells and have uncovered novel and fascinating mechanisms by which hES cells regulate cell death. We found that hES cells are highly sensitive to DNA damage, with all cells dying by 6 hours. A critical mediator of apoptosis in mammalian cells is Bax. In most cells, Bax is maintained in the cytosol in an inactive conformation and becomes activated only in response to apoptotic stimuli. Activated Bax then translocates to the mitochondria to induce cytochrome c release and caspase activation. Remarkably, we found that hES cells maintain Bax in its already active conformation. Surprisingly, active Bax was maintained at the Golgi rather than at the mitochondria, thus allowing hES cells to effectively minimize the risks associated with having pre-activated Bax. Our results show that after DNA damage, active Bax rapidly translocated from the Golgi to mitochondria by a p53-dependent mechanism. Thus, maintenance of Bax in its active form is a unique mechanism that can prime hES cells for rapid death, likely to prevent the propagation of mutations during the early critical stages of embryonic development. In this proposal, we will investigate this novel and unexpected mechanism by which apoptosis is regulated in hES cells. We will focus specifically on examining how Bax is maintained in an active state (Aim 1), determine how it localizes to the Golgi (Aim 2) and identify the molecular events triggered by DNA damage to induce the rapid translocation of active Bax from the Golgi to the mitochondria (Aim 3) in hES cells. These studies will undoubtedly uncover critical aspects of apoptosis regulation in cells and reveal key features of stem cell biology that can have significant impact for regenerative medicine.

DESCRIPTION (provided by applicant): Neurons have the capability to activate pathways that cause degeneration of either the entire cell by apoptosis or only the axons. Axon-specific degeneration is physiologically important as it allows neurons to remove excessive or misguided axon branches and permit plasticity in neuronal connections. However, exactly how a neuron can activate and compartmentalize this degenerative pathway to destroy its axon without putting the rest of the cell at risk is unclear. This is particularly interesting since recent studies idenified Bax, a key protein in the apoptosis pathway, to also regulate axon-specific degeneration. Our goal is to focus on the molecular intersection between apoptosis and axon-specific degeneration pathways. First, we established a microfluidic chamber-based model of sympathetic neurons where deprivation of nerve growth factor (NGF) from the axon compartment only induces axon-specific degeneration whereas NGF deprivation from the axon and soma compartments induces apoptosis. Second, we found substantial overlap between apoptosis and axon-specific degeneration but also identified distinct and unexpected differences. For example, while apoptosis required both Apaf-1 and Caspase-9 (Casp9), we found axon degeneration to require Casp9 but, surprisingly, not Apaf-1. Our results also show that neurons are exquisitely capable of selectively engaging apoptosis or axon-specific degeneration as mature neurons completely restrict apoptosis but remain permissive for axon degeneration. In this proposal, we will investigate several novel mechanisms by which neurons regulate axon-specific degeneration. We will focus specifically on determining how Bax is activated in this pathway (Aim 1), examining the mechanism by which Casp9 is activated independently of Apaf-1 (Aim 2), and probing the mechanism by which mature neurons selectively restrict apoptosis but remain permissive for axon degeneration (Aim 3). These studies will undoubtedly uncover critical aspects of how neurons utilize many of the same components for apoptosis and axon-selective degeneration but engage distinct mechanisms to allow precise spatial and temporal control over the activation of these pathways.

? DESCRIPTION (provided by applicant): The focus of our proposal is to evaluate whether Dicer inhibition is an effective strategy to selectively target medulloblastoma cells while sparing neurons. While the microRNA-dependent functions of Dicer are well known, recent studies have identified a novel, microRNA-independent function of Dicer in DNA damage repair. As maintenance of genomic integrity is critical for rapidly dividing cells that experience constitutiv replicative stress-induced DNA damage, we evaluated whether Dicer was essential for resolving such DNA damage in vivo in the context of developing cerebellum. In the absence of Dicer, the rapidly dividing cerebellar granule neuron precursors (CGNPs) accumulated DNA damage, which resulted in their degeneration. Remarkably, this degeneration was rescued by p53 deficiency, indicating that Dicer deficiency triggered the activation of the p53-mediated DNA damage pathway. In contrast to the high expression of Dicer in proliferating CGNPs, we found that Dicer is virtually undetectable in cerebellar granule neurons. These results suggest that unlike the proliferating CGNPs, Dicer may not be essential for survival in postmitotic neurons - a hypothesis we will test in a mouse model where we can conditionally delete Dicer selectively in the postmitotic cerebellar neurons. Importantly, we will also evaluate whether inactivation of Dicer could trigger cell death in medulloblastomas. Medulloblastomas are pediatric cerebellar tumors that arise from the aberrant and sustained proliferation of CGNPs beyond the developmental period. Medulloblastoma cells express Dicer, and we predict that, just as seen with the proliferating CGNPs during development, the proliferating medulloblastoma tumor cells also depend on Dicer for resolving endogenous DNA damage. We will test this hypothesis in two mouse models of medulloblastoma where we can readily assess the outcome of Dicer deletion on tumor growth as well as tumor regression. Together, these experiments critically evaluate the unexpected potential of Dicer as a therapeutic target for medulloblastoma with minimal neurotoxicity.

? DESCRIPTION (provided by applicant): Despite the identification of the main components of the mammalian apoptotic pathway, the differences in the regulation of apoptosis in various primary cells remain surprisingly unexplored. Our unique ongoing contribution to the apoptosis field has been to define how different cell types set the apoptosis threshold to optimally matche their physiological functions and adapt to changing environments. Indeed, we believe that new paradigms of apoptosis regulation remain to be discovered in physiologically distinct cell types. Our lab has discovered that the apoptotic pathway is highly restricted in postmitotic cells such as neurons, cardiomyocytes, and myotubes, as compared to mitotic cells such as fibroblasts. While a strict regulation of apoptosis is critical for the long-term survival of postmitotic cells, mitotic cells need to maintain their ability to activate apoptosis rapidly as they can be at continual risk of becoming cancerous. Therefore, cells must efficiently balance the need for having a primed apoptotic pathway versus the risks associated with cell death. In fact, we have seen this best exemplified in embryonic stem (ES) cells which engage mechanisms that both prime the apoptotic pathway for rapid death in response to DNA damage, while also engaging cell survival mechanisms in response to mitochondrial damage. Thus, ES cells appear to have an exquisite capability to respond to the specific damage stimuli with mechanisms that ensure both genomic integrity and optimal survival. In this MIRA proposal, we wish to use both targeted and broad integrative approaches to examine the distinct mechanisms of apoptosis regulation and define their physiological importance in health and disease. Our focus is on the two extremes of the apoptosis control we identified: 1) Mechanisms that resistant apoptosis and promote survival after mitochondrial damage (e.g. our findings that the E3 ligase PARC mediates the degradation of cytosolic cytochrome c), and 2) Mechanisms that prime cells for rapid apoptosis (e.g. our discovery that Bax is maintained in an active state in stem cells). We will use primary neurons and human embryonic stem (hES) cells, conduct innovative screens and examine disease implications in models of cancer and neurodegeneration. In particular, we are excited that the MIRA opportunity would enable our ambitious plans to use the powerful capability of hES cells to define how the apoptotic machinery undergoes dynamic changes with cellular differentiation.

Project Summary (30 lines of text) One of the underappreciated aspects of neuronal biology is that, as postmitotically-differentiated neurons become mature, they undergo dynamic changes to ensure that the mature nervous system is capable of long- term survival and function. Understanding these mechanisms that are critical for the long-term homeostasis of the adult brain is important as their dysfunction could increase the vulnerability of neurons to age-related neurodegeneration. We have identified miR-29 as a microRNA that is strikingly induced with neuronal maturation. miR-29 is not detectable during embryonic development, but its levels are induced more than 300 fold by 2 months and even greater by 6 months in the adult brain. In contrast to the high miR-29 levels that are maintained in the normal adult brains, miR-29 levels are markedly reduced in Alzheimer?s Disease patients. miR-29 is recognized to target many of the genes in the AD pathways including BACE1, ADAM10, PICALM, and NAV3. To evaluate the functional importance of miR-29, we recently generated mice in which miR-29 can be conditionally deleted. Mice deficient for miR-29, either in the whole body or in the brain, are born normal but then progressively decline, exhibiting neurological defects and early lethality. These results show that miR-29 has an essential function in the mature brain. Our hypothesis is that miR-29, while not needed for embryonic development, is physiologically important for maintaining long-term homeostasis in the adult brain. Reduction in miR-29 levels could therefore increase the vulnerability of mature neurons to become dysfunctional in the context of Alzheimer?s Disease. The overall focus of our proposal is to understand the endogenous mechanisms that maintain the very high levels of miR-29 in the normal brain, to critically examine the function on miR-29 is the adult brain, and to evaluate the therapeutic potential of miR-29 for Alzheimer?s Disease. Specifically, in Aim 1, we will test the hypothesis is that an increase in miR-29 transcription in mature neurons is a result of chromatin derepression. Importantly, we will also examine whether the substantial increase in miR-29 is a consequence of increased processing and stability in mature neurons. In Aim 2, we will focus on defining the molecular, cellular and behavioral consequences of deleting miR-29 in the adult brain. To evaluate the therapeutic potential of miR- 29 for Alzheimer?s Disease, we have also generated mice in which miR-29 can be conditionally overexpressed. Thus in Aim 3, we will examine whether overexpression of miR-29 is beneficial in the mutant APP knock-in mouse model of Alzheimer?s Disease. Overall, we are excited to be working on a molecule, miR-29, that has a unique and essential function in the mature brain. Our studies will help define its mechanisms of action as well as evaluate its therapeutic potential in the context of Alzheimer?s Disease.

DESCRIPTION (provided by applicant): This application requests the competing renewal of UNC's MSTP award. Our primary goal is to train an outstanding group of young men and women who are committed to become physician-scientists, fully capable of bridging the gap between basic science and clinical medicine. These individuals will go on to become the next generation of leaders in biomedical science, thereby making significant contributions to and improvements in human health. At the same time, they will become teachers and scholars not only at many of the best medical schools, but also at major research institutes and leading organizations in the biomedical and pharmaceutical industry. We hope to achieve our goal by identifying and then recruiting to UNC candidates from diverse backgrounds who bring with them a great variety of academic and research interests. Once here, they find a strong education in clinical medicine that is well integrated with superb research opportunities. Since the time of our last competing renewal, we have expanded our Leadership Team to include: Kim Rathmell, MD-PhD, the Director for Translational Science and Mohanish Deshmukh, PhD, the Director for Basic Science. In addition, based on the feedback that we received at the time of the last competing renewal of this MSTP grant, we have implemented several new initiatives designed to link the graduate school-based research activities of each student with relevant clinical experiences. Examples include: a) a Longitudinal Clinical Clerkship: this provides each student with clinical experiences that are related to and complement his/her research project; b) a monthly Clinical Case Conference: this is organized and directed by our senior MD-PhD students working in conjunction with Chief Residents from Medicine and Pediatrics; and c) a focus on the Clinical Relevance of Doctoral Dissertation: we are asking that each thesis committee include one clinician- scholar (often a physician-scientist), whose role is to ensure that the clinical relevance of the research is always at the forefront of the student's thinking. W believe that these additions have addressed the concerns of the last review panel and at the same time served to integrate the two phases of our training program. Over the past five years, our program has grown from 64 to 76 students, drawn to UNC from many of the best colleges and universities all across the USA. Their academic credentials are superior: this year's incoming class, for example, had a mean GPA of 3.75 and a mean MCAT of 37.6. Once here, our students are performing at a high level in both the classroom and the laboratory. They are pursuing their graduate training in 20 individual departments and curricula representing the Schools of Pharmacy, Public Health, Medicine, and the College of Arts and Sciences. They are receiving a high percentage of honors, publishing on average four manuscripts, successfully competing for a variety of awards and independent funding (e.g., 26 of our students hold F30 awards from the NIH), and completing the dual degree program in eight years. After completing their post-doctoral training, many of our graduates are becoming faculty members at many excellent institutions.

Project Summary/Abstract (30 lines of text) Despite our knowledge about DNA damage responses, we know significantly less about the cellular pathways activated selectively with mitochondrial DNA (mtDNA) damage. In fact, when cells are exposed to DNA damaging drugs, the responses observed are likely a consequence of damage to both the nuclear DNA and mtDNA. However, separating the outcome of nuclear DNA damage versus mtDNA damage is difficult because of the challenges of exposing only the mitochondria and not the nucleus to DNA damage in cells. To selectively expose the mitochondria but not the nucleus to DNA damaging drugs, we utilized primary neurons as their cell body (containing the nucleus) and axons (containing mitochondria but not the nucleus) are spatially distinct. We use microfluidic chambers to culture the neurons, which allowed us to expose only the axons and not the cell bodies to DNA damaging drugs such as cisplatin. Addition of cisplatin exclusively to the axons in microfluidic chambers induced mtDNA damage in axonal mitochondrial without any DNA damage to the nucleus. Importantly, cisplatin addition induced widespread axon degeneration. No degeneration was seen in the cell body or neuronal regions that were not exposed to cisplatin. Similar selective degeneration of axons was also observed when axons were exposed to other DNA damaging drugs such as the nucleoside analog d4T or the topoisomerase I inhibitor camptothecin. In thisR21 proposal, we will focus on defining the molecular mechanism of axon degeneration induced with mtDNA damage. While nuclear DNA damage is recognized to activate a p53-depedent apoptotic cell death pathway, virtually nothing is known about the specific cell degeneration pathway activated with mtDNA damage. Thus, in Aim 1 we will investigate whether key proteins of the apoptotic pathway are required for axon degeneration in response to mtDNA damage. Specifically, we will evaluate mtDNA damage-induced axon degeneration in wildtype neurons or neurons deficient in p53, Bax, Apaf-1 Caspase-9 or Caspase-3. In addition to apoptosis, axon degeneration has been studied in the context of pruning and axotomy-induced Wallerian degeneration. Thus, in Aim 2, we will examine whether axon degeneration after mtDNA damage is mediated by either the pruning (using Caspase-6-deficient neurons) or Wallerian (using Sarm-1-deficient neurons) axon degeneration pathways. Our focus in Aim 3 is on mitophagy as damaged mitochondria are recognized to be targeted for degradation via Parkin-mediated degradation. While mitophagy is generally beneficial to neurons, excess mitophagy could also be detrimental, particularly in situations of acute mitochondrial damage. We will utilize Parkin-deficient neurons to critically evaluate whether inhibition of mitophagy accelerates or reduces the kinetics of axon degeneration with acute mtDNA damage. Our approach of inducing mtDNA damage in axons using the neuronal microfluidic chamber model has broad relevance, as it will help define the nuclear DNA damage-independent mechanisms of degeneration.

Project Summary/Abstract Neurons have the capability to activate pathways that cause degeneration of either the entire cell by apoptosis or only the axons. Physiological axon-specific degeneration, also known as pruning, is important as it allows neurons to remove excessive or misguided axon branches and permit plasticity in neuronal connections. However, exactly how a neuron can activate and compartmentalize this pathway to degenerate its axon without putting the rest of the cell at risk is unclear. This is particularly interesting since recent studies identified Bax and caspases, key proteins in the apoptosis pathway, to also regulate axon pruning We are using a microfluidic chamber-based model of sympathetic neurons where deprivation of nerve growth factor (NGF) can induce either apoptosis (when NGF is deprived from both soma and axon compartments) or axon pruning (when NGF is deprived from only the axon compartment). Using this model, we identified substantial overlap but also distinct differences between the apoptosis and axon pruning pathways. Specifically, we found that while caspase-9 (Casp9) is required for both pathways, Casp9 activation is dependent of Apaf-1 during apoptosis but, surprisingly, independent of Apaf-1 during axon pruning. These results were unexpected and point to a novel mechanism of Casp9 activation during axon pruning. In this proposal, our goals are to examine specific aspects of how Casp9 is activated during axon pruning. In Aim 1, we will focus on Bax function during axon pruning. Since Apaf-1 is not needed for axon pruning, we propose that the essential function of Bax during axon pruning is not the release of cyt c (which activates Apaf- 1 in the context of apoptosis), but instead is the release of the Smac, an inhibitor of XIAP, from mitochondria. In Aim 2, we will examine how Casp9 is activated during pruning. Our focus is on identifying key features of Casp9 function that are important for axon pruning. Additionally, we will examine whether components of a dependosome-like complex are important for activating Casp9 during axon pruning. These studies will help uncover critical aspects of how neurons utilize many of the same components for apoptosis and axon pruning yet with distinct differences.

Project Summary/Abstract Neurons are capable of activating pathways that induce either the degeneration of the entire cell by apoptosis or to selectively degenerate only the axons by pruning. While the main components of the caspase activating machinery during apoptosis and pruning have been identified, a fundamental question of whether the apoptotic machinery is activated throughout the neuronal soma and axons, or is spatially restricted, during these events remains unknown. This question is most relevant in the context of pruning where one predicts the apoptotic machinery to be localized to the targeted axons undergoing degeneration. However, we were surprised to find an unexpected spatial restriction of caspase activation even during apoptosis, where we found these to be restricted primarily to the soma, even though both soma and axons degenerate. In this proposal, we will mechanistically examine the spatial localization of the apoptotic machinery in neurons during apoptosis and pruning. We will utilize neurons cultured in microfluidic chamber devices to allow for the spatial segregation and manipulation of neuronal somas and axons. Our hypothesis is that during apoptosis and pruning, the restricted caspase activity causes the ?physiological axotomy? of axons, activating the Sarm1-mediated axotomy pathway of axon degeneration. The concept that the developmental pathways of apoptosis and pruning can cause axotomy is novel because these pathways were considered to be distinct from the injury-induced axotomy pathway. In Aim 1, we will define the spatial restriction of the apoptotic machinery in neurons during apoptosis and axon pruning. In Aim 2, we focus on examining the function of Sarm1 during apoptosis and axon pruning. In Aim 3, we will investigate the Sarm1-deficient mice for pruning defects in vivo and evaluate if these mice exhibit behavioral deficits. This project opens exciting areas of research not only because of its new concepts for apoptosis and pruning, but also because it brings into focus a developmental function of Sarm1 that is beyond its recognized role in axon injury.

Project Summary/Abstract Neurons are capable of activating pathways that induce either the degeneration of the entire cell by apoptosis or to selectively degenerate only the axons. Physiological axon-specific degeneration, known as axon pruning, is important as it allows neurons to remove excessive or misguided axons and permit plasticity in neuronal connections. Aberrant pruning is observed in several neurodegenerative diseases, including Alzheimer?s Disease (AD). However, exactly how neurons activate this pathway to degenerate axons in physiological or pathological situations of AD is unclear. We have investigated the apoptosis and axon pruning pathways in a microfluidic chamber-based model utilizing sympathetic neurons. Upon nerve growth factor (NGF) deprivation, these neurons can induce either apoptosis (when NGF is deprived from both soma and axon compartments) or axon pruning (when NGF is deprived from only the axon compartment). Our research has identified substantial overlap but also distinct differences between the apoptosis and axon pruning pathways. For example, while caspase-9 (Casp9) and caspase-3 (Casp3) are required for both pathways, their activation is dependent on the apoptosome during apoptosis but is surprisingly independent of the apoptosome during axon pruning. While investigating the mechanism by which caspases are activated during pruning, we unexpectedly found that the inflammasome pathway plays an important function in axon pruning. Inflammasomes have been studied primarily in immune cells in the context of pathogen signaling. These are multi-protein complexes formed in response to pathogenic or danger stimuli, which result in activation of the proinflammatory caspase, caspase-1 (Casp1). Strikingly, we found that Casp1 and NLRP1 (a key component of a particular inflammasome) are both essential for axon pruning. These results are surprising as axon pruning does not involve pathogen exposure. In this proposal, we will identify the specific inflammasome pathway components that are critical for axon pruning and conduct mechanistic experiments to define this novel function of NLRP1 in neurons. In Aim 1, we will define the specific inflammasome proteins that are essential for axon pruning, and determine where they act in the known pruning pathway. In Aim 2, we will define the role of IL-1?/IL-18 in axon pruning. Our focus in Aim 3 will be to conduct mechanistic experiments to examine how NLRP1 is activated in the context of axon pruning. Importantly, in Aim 4 we will focus on AD and investigate whether the pathological degeneration of synapses and axons in AD are mediated by NLRP1. We will examine this in a microfluidic model of A?-induced axon degeneration in vitro as well as in a mouse model of AD in vivo where we will examine if NLRP1 deficiency reduces AD pathology and behavioral defects. This project opens an exciting new avenue of research into this unexpected function of the NLRP1 inflammasome in neurons.