Bingwei Lu - US grants

DESCRIPTION: Asymmetrical cell division and protein localization is a fundamental process in eukaryotic and prokaryotic cells. The neural precursor cell division in Drosophila serves as a model system for studying the mechanisms of asymmetric cell division in general. In this system, both genes responsible for establishing cellular polarity and genes playing specific roles in direct protein localization are required for the asymmetric distribution of cell fate determinants, such as Numb. The goal of this proposal is to address the function of I3, a candidate component of the cellular machinery that is localizing Numb. The consequences of loss-or gain-of I3 function will be assessed by isolating I3 mutants and by ectopically expressing I3. The relationship between I3 and genes known to participate in asymmetric cell division will be established by molecular epistasis analysis. Structure-function analysis will uncovered the domain responsible for the asymmetric localization of I3 and test the functioning of Numb-I3 interaction. By isolating proteins that associate with I3, additional components involved in the process will be identified. This will provide us with a broader view of how cellular symmetry is established and how asymmetric protein localization is accomplished. It is expected that studying asymmetric cell divisions in Drosophila will advance our understanding of stem cell divisions in humans and lead to therapeutic inventions in the treatment of cancer and neurodegenerative diseases.

[unreadable] DESCRIPTION (provided by applicant): Neural stem cells (NSCs) play critical roles in generating neurons and glia during both embryonic development and adult life. Aberrant NSC behavior could lead to neurological disorders, especially those with developmental etiology, and brain tumors. Drosophila neuroblast provides an excellent model for understanding fundamental aspects of NSC regulation in vivo. The long-term goal of this proposal is to elucidate mechanisms regulating the self-renewal and differentiation of NSCs through combined cell biological, genetic, and molecular analyses. Drosophila neuroblasts undergo stem cell-like self-renewing asymmetric divisions. In each division, one daughter cell remains as a stem cell while the other is committed to differentiation. Factors controlling stem cell self-renewal and differentiation are distributed in a polarized fashion and the mitotic spindles are oriented with respect to this polarity axis, ensuring proper segregation of these important factors between the two daughter cells. Numb is such a factor that is segregated to the differentiating daughter cell during neuroblast division, where it inhibits the renewal of neuroblast cell fate. The asymmetric segregation of Numb requires Partner of Numb (Pon). In the last four years we have focused on identifying molecules that impinge on Pon to regulate Numb and those that regulate mitotic spindle orientation. We have learned that a number of mitotic kinases directly act on Pon and Numb. This raises the interesting possibility that during NSC asymmetric division, a series of mitotic kinases act in concert to ensure the faithful segregation of key cell fate determinants to the appropriate daughter cells. We propose to test this hypothesis. We have also learned that a number of tumor suppressors and kinases are involved in regulating mitotic spindle orientation. We propose to investigate the biochemical and genetic relationships between these signaling molecules and to place them into a pathway. We also propose to assess the contribution of the Pon/Numb pathway and the spindle orientation pathway in controlling NSC self-renewal and to identify new players involved in this process. Given the evolutionary conservation of the molecules studied in this proposal and the conservation of mechanisms underlying NSC asymmetric division, knowledge to be gained from this study shall provide important insights into the understanding of mammalian NSC regulation in vivo and ultimately treating diseases originated from dysfunctional NSCs. [unreadable] PUBLIC HEALTH REVELANCE: Neural stem cells are multi-potent cells that generate the diverse cell types in our brain. The goal of this proposal is to achieve a mechanistic understanding of the basic properties of these special cells and how disease may arise when neural stem cells behave abnormally. Accomplishment of the proposed aims will ultimately help understand and treat a number of neurological disorders originated from aberrant neural stem cells, such as neurodegenerative diseases and brain tumors. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Tuberous sclerosis complex (TSC) is a human syndrome characterized by widespread development of benign tumors in multiple tissues, with lesions in the brain causing the most debilitating symptoms such as seizures and mental retardation. Germline mutations in either TSC1 or TSC2 tumor suppressor genes cause this syndrome. Recent genetic studies in Drosophila and biochemical studies in mammalian cells have suggested that TSC1 and TSC2 negatively regulate the target of rapamycin (TOR) signaling pathway to control cell growth. Abnormal activation of TOR signaling may thus underlie the pathogenesis of TSC and other benign tumor syndromes. TOR signaling appears to serve as a crucial integration point that coordinates cell growth and survival with energy and nutrient conditions. Little is known about the molecular mechanisms by which diverse environmental or physiological signals feed into the TSC/TOR signaling pathway to control cell growth and survival. In our preliminary studies of a Drosophila homologue of human DJ-1 gene, which is associated with familial Parkinson's disease, we have found that inhibition of DJ-1A leads to impaired TOR signaling and cell death and that DJ-1A genetically interacts with certain components of TSC/TOR pathway to promote cell survival. These results implicate DJ-1A as a novel regulator of TSC/TOR signaling. The goal of this proposal is to achieve a mechanistic understanding of the interaction between DJ-1A and the TSC/TOR pathway in promoting cell survival. The proposed genetic and biochemical analyses will provide novel insights into the regulation and function of TSC/TOR signaling. Further studies along this direction could solidify a fundamental role for TSC/TOR signaling in promoting cell survival under pathological conditions. These studies could implicate a common biochemical pathway in the pathogenesis of TSC and Parkinson's disease and offer one avenue for elucidating the molecular events that cause the development of brain lesions in TSC patients. Further studies could lead to the identification of new therapeutic targets and ultimately help develop rational mechanism-based treatment strategies that target TSC brain lesions. [unreadable] [unreadable] These studies could implicate a common pathway in the pathogenesis of TSC and Parkinson's disease. Further studies could lead to the identification of new therapeutic targets and ultimately help develop mechanism-based treatment strategies that target TSC brain lesions which cause the most devastating symptoms of the disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Genetic studies have revealed high level conservation of genes and pathways controlling the formation and patterning of muscle fibers during vertebrate and invertebrate development. Much less is known about the molecular mechanisms that help maintain the integrity and function of the different types of muscles after they are fully developed. Failure in the maintenance of muscle integrity and function can lead to debilitating muscle diseases. The goal of this proposal is to use Drosophila genetics to identify genes and pathways that control muscle maintenance. We have recently found that inactivation of the Drosophila homologue of human Pten-induced kinase 1 (Pink1) results in relatively selective degeneration of the indirect flight muscle (IFM), an extremely fast and metabolically active muscle. Muscle degeneration is preceded by dysfunction and degeneration of the mitochondria. Our genetic and biochemical studies indicate that Pink1 acts in the same pathway as Parkin, an E3 ubiquitin ligase also linked to mitochondria and muscle maintenance. We propose to carry out detailed biochemical and genetic analyses to understand the in vivo relationship between Pink1 and Parkin. In preliminary genetic interaction studies we have found that genes involved in regulating mitochondrial physiology can modify Pink1 mutant phenotypes. We propose to understand how these genes and Pink1 interact at the molecular and cellular levels. We have also identified genes that can enhance or suppress Pink1-associated muscle degeneration in an unbiased pilot genetic screen. We propose to expand this genetic modifier screen to systematically survey the Drosophila genome for new genes that function in the Pink1/Parkin pathway of mitochondria and muscle maintenance. Key genes will be selected and subjected to in depth phenotypic and molecular characterization and organized into genetic pathways. Understanding the function of the genes studied in this project will provide crucial insights into the fundamental mechanisms linking mitochondrial function with muscle maintenance. This will ultimately contribute to the treatment or prevention of degenerative muscle diseases, especially those with mitochondrial etiology. Given the association of Pink1 and Parkin to Parkinson's disease and the involvement of mitochondrial dysfunction in a host of neurodegenerative diseases, results to be obtained from this study will also be highly relevant to the maintenance of nervous system integrity. PUBLIC HEALTH RELEVANCE. The goal of this proposal is to understand the role of Pink1 protein in maintaining mitochondrial function and skeletal muscle integrity. Information to be gained from this study will help understand and ultimately treat muscle-related diseases, especially those with mitochondrial etiology, such as mitochondrial myopathy. Since Pink1 is also involved in Parkinson's disease, the information will also provide insights for the understanding and treatment of this devastating brain disorder.

DESCRIPTION (provided by applicant): Mitochondria play essential roles in normal neuronal physiology, from energy production to Ca2+ buffering to synaptic differentiation and plasticity. The intracellular distribution of mitochondria needs to be precisely matched to the demand for these organelles, a task particularly difficult for neurons due to their highly polarized morpholog and their dynamic patterns of neuronal activity and synaptic plasticity in vivo. Because abnormal mitochondrial distribution and function has been consistently observed at early stages of neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD) as well as neuropsychiatric disorders, understanding the genetic control of mitochondrial distribution in neurons has assumed a high priority in neuroscience research. Unfortunately, progress in this area has been impeded by the lack of identified regulatory molecules governing this process. In the proposed project, we aim to define the role of PAR-1 (partitioning defective 1), an evolutionarily conserved Ser/Thr kinase, in the regulation of neuronal mitochondrial distribution. PAR-1 was initially identified as a gene required for the asymmetric cell division in early C. elegans embryos. Work from our lab and that of others has established a critical role for PAR-1 in regulating synaptic structure and function in Drosophila and mammals. Our most recent work shows that PAR-1 plays an important role in directing neuronal mitochondrial distribution. Additional studies indicate that PAR-1 genetically and physically interacts with mitochondrial rho GTPase (Miro), a conserved key component of the mitochondrial transport machinery, and that PAR-1 regulates the GTPase activity of Miro as well as the interaction between Miro and the mitochondrial fusion regulator mitofusin (Mfn). These findings led logically to the central hypothesis of the current application: that the PAR-1/Miro axis functions as a novel regulatory node through which diverse signals can impact mitochondrial distribution, and that deregulated PAR-1/Miro signaling contributes to the mitochondrial maldistribution and the ensuing synaptic dysfunction and eventual neurodegeneration as occurring in diseases. This hypothesis will be tested by determining the mechanism of how PAR-1/Miro signaling regulates mitochondrial distribution in Drosophila (Aim 1); by testing the effect of restoring PAR-1/Miro-directed mitochondrial distribution on the disease phenotypes of Drosophila models of AD (the Abeta-42 model) and PD (the LRRK2-G2019S model) (Aim 2); and by testing the effect of restoring PAR-1/Miro-directed mitochondrial distribution on the disease phenotypes of patient-specific, Abeta-42 and LRRK2-G2019S-related human neuronal models of AD and PD (Aim 3). Successful completion of these aims will be facilitated by innovative methods and strategies for visualizing and manipulating mitochondrial distribution in vivo in Drosophila and in human neuronal models of AD and PD. We expect that the information to be generated from this project will be fundamental to basic neuroscience research and of high clinical relevance.

DESCRIPTION (provided by applicant): Achieving homeostasis within neural stem cell (NSC) lineages is essential for nervous system development and maintenance. It requires an exquisite balance between NSC self-renewal and differentiation. The molecular and cellular mechanisms underlying the control of NSC homeostasis remain poorly understood. Elucidation of these mechanisms will provide novel insights into the development and maintenance of the nervous system as well as offer the keys to molecularly targeted therapy for diseases resulting from NSC homeostasis failure, including brain tumors and neurodevelopmental, psychiatric, and neurodegenerative disorders. We propose to elucidate the basic mechanisms underlying the genetic control of NSC homeostasis, using Drosophila larval brain type II neuroblasts (NBs) as a model. Drosophila NBs have been instrumental in discovering signaling molecules such as Numb and Notch, and cellular mechanisms such as asymmetric cell division, that are centrally involved in NSC homeostasis. Like mammalian NSCs, fly type II NBs generate transit-amplifying intermediate progenitors (IPs), which help to generate a vast number of differentiated progenies. Notch signaling is critical for maintaining the stemness of type II NBs. Inhibition of Notch signaling results in NB fate not being properly maintained, whereas aberrant Notch activation causes ectopic NB formation and brain tumorigenesis. The function of Notch in regulating NSC homeostasis appears to be conserved in mammals. However, there is much to be learned about the mechanisms of action of Notch and its in vivo relationship with Numb, which remains enigmatic and controversial. We have found that canonical Notch signaling is necessary but not sufficient for Notch-directed NSC regulation and that a novel non-canonical Notch signaling pathway is also involved. Our main hypothesis is that non-canonical Notch signaling acts coordinately with canonical Notch signaling to mediate distinct aspects of NSC homeostasis control, and that Numb regulates both of these two pathways. Many fundamental questions are raised: What cellular programs do the canonical and the non-canonical pathways regulate? What are the key molecular targets of these pathways? Can we recapitulate the effect of Notch by manipulating these key targets? What roles does Numb play in these two pathways? Three specific aims will help address these questions. Aim 1 will genetically and biochemically elucidate a non-canonical Notch signaling pathway that regulate NSC homeostasis. Aim 2 will test the hypothesis that the non-canonical Notch pathway and the canonical pathway act coordinately to maintain NSC homeostasis. Aim 3 will test the hypothesis that Numb acts in a novel protein complex to regulate key downstream mediators in the canonical and non-canonical Notch pathways. Upon successful completion of these Aims, we will generate new mechanistic insights into the control of NSC homeostasis by Notch and Numb. We anticipate that this will open up entirely new directions for studying the fundamental roles of Numb and Notch in NSC biology and cancer biology.

DESCRIPTION (provided by applicant): Aging is a fundamental biological process that occurs in all eukaryotic organisms. Despite intensive research, the molecular and cellular mechanisms underlying this complex process remain poorly understood. Altering mitochondrial function and nutrient composition in diets are two life span- extension conditions that are drawing considerable interests in the aging field due to their occurrence in all model organisms tested so far. Changes in mitochondrial morphology and function have been intimately associated with aging in diverse organisms. Moreover, genetic and dietary manipulations that extend life span in various species are dependent on normal mitochondrial function. Paradoxically, certain perturbations of mitochondrial function can also extend life span in yeast, worms, flies, and mammals. Thus, the exact role of mitochondria in life span regulation remains enigmatic. Recent studies have also implicated epigenetic regulators in controlling life span in multiple organisms. Despite the strong evidence supporting the importance of mitochondrial and epigenetic regulations in life span determination, the relationship, if any, between these two longevity-regulating axes in the aging process has not been established in metazoans. Our preliminary studies in the model organism Drosophila have uncovered a novel connection between mitochondrial function and epigenetic modifications. We found that the functional status of mitochondria can directly affect histone H3 acetylation, an epigenetic regulation that can strongly influence gene transcription in the nucleus. Our genetic studies strongly implicated the histone acetyltransferase GCN5 as a key enzyme mediating mitochondria-induced H3 acetylation. Moreover, our results implicated the involvement of target of rapamycin complex-2 (TORC2) in this process. These findings led logically to our central hypothesis that mitochondrial and dietary alterations (e.g., dietary restriction-DR) extend life span by impinging on GCN5-mediated acetylation of histone H3 to activate the transcription of longevity promoting genes, and that this process is mediated by TORC2. The proposed work will employ the powerful tools uniquely available in Drosophila to test this novel hypothesis concerning the relationship between mitochondrial and epigenetic changes in promoting longevity. There are two specific Aims in this proposal. In Aim 1, we will examine the role and tissue- specific requirement of GCN5 in mitochondrial and dietary alteration-induced life span extension. In Aim 2, we will determine the relationship between GCN5 and TORC2 in mediating the effects of mitochondrial and dietary alterations on life span extension. These studies will offer novel insights into the mechanisms of a prolongevity signaling network underlying the effects of mitochondrial metabolism and diet on life span by elucidating the roles of TORC2 and GCN5 in the process. This will ultimately offer new ways to interfere with the aging process and provide novel therapeutic strategies to treat a battery of age-related diseases.

DESCRIPTION (provided by applicant): Genetic studies in model organisms have provided tremendous insights into neural development and revealed surprising similarities between vertebrates and invertebrates in the genes and pathways controlling the patterning and wiring of the nervous system. Compared to neural development, much less is known about the molecular and cellular mechanisms that help maintain the integrity and function of the diverse differentiated neurons after they are fully developed and integrated into neural circuits. It is expected that elucidation of the mechanisms central to neuronal maintenance in model organisms will inform similar processes in humans, impairments of which underlie various neurodegenerative conditions such as Alzheimers disease and Parkinsons diseases, for which there is currently no effective treatment. Drosophila has served as an excellent model system to elucidate the signaling network that directs mitochondrial quality control, a multifaceted process encompassing fission/fusion dynamics, transport, and autophagy (mitophagy). This mitochondrial quality control process is crucially important for the structural and functional integrity of dopaminergic neurons, the cell types that are lost to Parkinsons disease. Our recent genetic studies have revealed novel roles of the conserved target of rapamycin signaling complexes (TORC1 and TORC2) in regulating mitochondrial function and maintaining dopaminergic neuron integrity, although paradoxically TORC1 and TORC2 exhibit opposite effects in this process. The goal of this proposal is to use molecular genetic, genomic, biochemical, and cell biological tools available in Drosophila to decipher the mechanisms of action of TORC1 and TORC2 in mitochondrial regulation, in an effort to understand in molecular terms how mitochondrial abnormality arises and how it impacts neuronal integrity in age-related neurodegenerative disease conditions. The hypothesis to be tested is that TORC1 and TORC2 play central roles in dopaminergic neuron maintenance by directing distinct aspects of mitochondrial regulation, with TORC2 regulating mitochondrial quality control whereas TORC1 regulating mitochondrial respiratory chain complex biogenesis through translational regulation. Key findings from the fly studies will be validated in patient-derived, dopaminergic neuron-based disease models. Greater understanding of the functions of the genes to be studied in this project will provide novel insights into the fundamental mechanisms linking mitochondrial regulation to neuronal maintenance. This will ultimately contribute to the treatment of a host of neurodegenerative conditions associated with mitochondrial dysfunction.

Project Summary: An intersection between neural development and neurodegeneration processes is increasingly being recognized in many neurological diseases, including neurodevelopmental and psychiatric diseases such as schizophrenia. Understanding the molecular basis underlying the connection between these two seemingly disparate processes promises to provide fundamental insights into the logic and principles of neuronal development and maintenance. Mitochondria are dynamic and complex organelles with essential roles in many aspects of biology, from energy production and intermediary metabolism to apoptosis and Ca2+ buffering. Mitochondrial dysfunction has been observed early on in neurodegenerative diseases such as Parkinson?s disease and in diseases suspected to have both neurodevelopmental and neurodegenerative components such as schizophrenia. How mitochondrial dysfunction arises in the disease process, and the exact roles of mitochondria in neuronal development and maintenance are not well understood. In this project we propose to test the hypothesis that the tRNA Splicing Endonuclease (TSEN) complex acts through regulation of mitochondrial function to influence neuronal development and maintenance. Genetic mutations in TSEN have been linked to Pontocerebellar Hypoplasia (PCH), a rare congenital disorder characterized by neurodevelopmental deficits as well as neurodegeneration. The molecular function of TSEN and the cellular mechanism of TSEN dysfunction on neuronal development or maintenance are poorly understood. Based on compelling preliminary results, we hypothesize that the TSEN complex critically regulates the metabolism and/or translation of nuclear encoded respiratory chain component (nRCC) mRNAs to maintain mitochondrial function. To test this hypothesis, we propose to achieve two Specific Aims. In Aim 1, we seek to firmly establish the mitochondrial basis of TSEN-associated disease pathogenesis. In Aim 2, we intend to determine the molecular mechanisms by which the TSEN complex regulates mitochondrial function. Drosophila TSEN models will be extensively used in this project. Our lab is highly experienced in the use of Drosophila as a model to dissect cellular signaling pathways commonly involved in regulating nervous system development and maintenance, in an effort to gain novel insights into the pathogenesis of neural developmental and degenerative diseases. We are particularly experienced in studying the role of mitochondria in these fundamental processes. Using the techniques we have developed and the expertise we have acquired, we aim to generate the first in vivo evidence that TSEN critically regulates mitochondrial function. We anticipate that results from this study will set the stage for future mechanistic studies of TSEN regulation of mitochondria using Drosophila as a model, which promises to inform the understanding and treatment of other neural developmental/degenerative disorders with similar mitochondrial etiologies.

Project Summary Neurons exhibit highly polarized morphology and make intricate synaptic connections with other cells in the body. The strength of such connections responds to neuronal activity and can be modulated at individual synapse level. Such unique features of polarized morphology, intricate connectivity, and functional plasticity necessitate precisely controlled gene expression in neurons. Translational control has emerged as a critical regulatory mechanism that confers spatiotemporal precision to neuronal gene expression. In addition, by influencing energy expenditure in cells - considering that protein synthesis is a very energy-consuming process, and by modulating levels of misfolded or aggregated proteins, translational control is intimately linked to energy metabolism and proteostasis, two processes essential for neuronal maintenance. It is thus expected that translational control will assume particular importance in normal neurobiological processes such as synaptic plasticity, learning, and memory, and in the pathogenesis of neurological disorders. However, compared to other regulatory mechanisms of gene expression such as transcriptional control, our understanding of the mechanism and function of translational control in health and disease is lagging behind. In the proposed project, we aim to define the mechanism of action of LRRK2 (leucine-rich repeat kinase 2), a gene most frequently mutated in familial and sporadic Parkinson's disease, in the regulation of mRNA translation in disease-relevant human dopaminergic neurons. Based on strong preliminary studies, we hypothesize that LRRK2 participates in the translational control of mRNAs in human dopaminergic neurons by acting through distinct substrates and/or effectors to regulate translation at the initiation and elongation steps. To test this hypothesis, we will use human induced dopaminergic neurons (iDNs) reprogrammed from patient fibroblasts and the powerful CRISPR/Cas9 genome editing technique to determine the mechanisms and function of translation initiation and elongation control by LRRK2 (Aim 1), and to profile the molecular signatures of LRRK2-regulated mRNAs and proteins (Aim 2). Execution of this project will be facilitated by innovative technologies and strategies for studying translational control in reprogramming-derived human neurons. Successful completion of this project will provide new insights into the biology and pathobiology of LRRK2 and validate a new platform for mechanistic studies of human neurological diseases using patient-derived neurons and CRISPR/Cas9. The information to be generated from this project is therefore expected to be fundamental to basic neuroscience research and of high clinical relevance.

Project Summary: An intersection between neural development and neurodegeneration processes is increasingly being recognized in many neurological diseases, including neurodevelopmental and psychiatric diseases such as schizophrenia. Understanding the molecular basis underlying the connection between these two seemingly disparate processes promises to provide fundamental insights into the logic and principles of neuronal development and maintenance. Mitochondria are dynamic and complex organelles with essential roles in many aspects of biology, from energy production and intermediary metabolism to apoptosis and Ca2+ buffering. Mitochondrial dysfunction has been observed early on in neurodegenerative diseases such as Parkinson?s disease and in diseases suspected to have both neurodevelopmental and neurodegenerative components such as schizophrenia. How mitochondrial dysfunction arises in the disease process, and the exact roles of mitochondria in neuronal development and maintenance are not well understood. In this project we propose to test the hypothesis that the tRNA Splicing Endonuclease (TSEN) complex acts through regulation of mitochondrial function to influence neuronal development and maintenance. Genetic mutations in TSEN have been linked to Pontocerebellar Hypoplasia (PCH), a rare congenital disorder characterized by neurodevelopmental deficits as well as neurodegeneration. The molecular function of TSEN and the cellular mechanism of TSEN dysfunction on neuronal development or maintenance are poorly understood. Based on compelling preliminary results, we hypothesize that the TSEN complex critically regulates the metabolism and/or translation of nuclear encoded respiratory chain component (nRCC) mRNAs to maintain mitochondrial function. To test this hypothesis, we propose to achieve two Specific Aims. In Aim 1, we seek to firmly establish the mitochondrial basis of TSEN-associated disease pathogenesis. In Aim 2, we intend to determine the molecular mechanisms by which the TSEN complex regulates mitochondrial function. Drosophila TSEN models will be extensively used in this project. Our lab is highly experienced in the use of Drosophila as a model to dissect cellular signaling pathways commonly involved in regulating nervous system development and maintenance, in an effort to gain novel insights into the pathogenesis of neural developmental and degenerative diseases. We are particularly experienced in studying the role of mitochondria in these fundamental processes. Using the techniques we have developed and the expertise we have acquired, we aim to generate the first in vivo evidence that TSEN critically regulates mitochondrial function. We anticipate that results from this study will set the stage for future mechanistic studies of TSEN regulation of mitochondria using Drosophila as a model, which promises to inform the understanding and treatment of other neural developmental/degenerative disorders with similar mitochondrial etiologies.

Project Summary Age-related neurodegenerative diseases such as Parkinson's disease (PD) impose tremendous socioeconomic burdens due to the lack of disease-modifying treatment options. Mitochondrial dysfunction is intimately linked to neurodegenerative diseases. How mitochondrial abnormalities arise and how they relate to other features of neurodegenerative diseases such as proteostasis failure, Ca2+ dyshomeostasis, and neuroinflammation are poorly understood. Dynamic control of the structure, function, and distribution of mitochondria is also essential for normal neuronal function, a requirement necessitated by the highly polarized shape and unique physiology of neurons. Despite intensive efforts, many fundamental questions remain regarding the mechanisms linking mitochondrial regulation to neuronal maintenance. Pten-induced kinase 1(PINK1) and Parkin (encoding an E3 ubiquitin ligase), two genes associated with familial PD, have defined a genetic pathway important for mitochondrial and neuronal maintenance in flies and mammals. Identification of this pathway offers a much-needed entry point to understand the regulation of mitochondrial function in response to neuronal activity and metabolic needs, and to decipher the mechanistic link between mitochondrial dysfunction and other pathological hallmarks of disease. Our genetic studies revealed that PINK1/Parkin directs an interconnected mitochondrial quality control (MQC) process important for the maintenance of dopaminergic (DA) neurons. The multifaceted MQC process encompasses translational control of respiratory chain complex (RCC) biogenesis, mitochondrial fission/fusion dynamics, transport, and removal of defective mitochondria by autophagy (mitophagy). In the past funding period we have shown that the conserved target of rapamycin complexes (TORC1 and TORC2) act as important mediators of PINK1-directed MQC. One exciting finding from our investigation is that the PINK1/mTORC2 pathway exerts translational control of nuclear encoded RCC (nRCC) mRNAs. The goal of this proposal is to move away from the status quo of mitophagy-centric focus of PINK1-directed MQC by focusing on the newly discovered translational control function of PINK1/mTORC2 signaling. We will use a unique combination of molecular genetic, genomic, cell biological, and biochemical tools, and move between in vivo fly models and in vitro induced DA neuron (iDN) models. Our central hypothesis is that PINK1/mTORC2 signaling regulates DA neuron function and survival through ribosome-associated co-translational quality control (RQC) of select nuclear-encoded mitochondrial mRNAs, thus mechanistically linking mitochondrial function to protein homeostasis. We propose to elucidate how the RQC pathway mediates the effects of PINK1/mTORC2 on mitochondrial regulation and DA neuron maintenance (Aim 1), and dissect the molecular mechanism of RQC regulation by PINK1/mTORC2 signaling in both Drosophila models and patient-derived iDN models (Aim 2). These studies will significantly advance our understanding of how PINK1/mTORC2 signaling regulates DA neuron homeostasis, shed light on the poorly understood phenomenon of neuronal vulnerability to RQC failure, and potentially lead to novel and rational therapy for PD and other neurological disease conditions.

Project Summary Muscle is a contractile tissue that generates forces and motions vital for animal survival. The molecular and cellular mechanisms governing its structural and functional integrity are not well understood. Selective dysfunction and degeneration of neuromuscular tissues have been observed in disease conditions featuring mitochondrial abnormality, emphasizing the particular importance of mitochondria to the functionality and integrity of muscle tissues. Mitochondria play important roles in cellular bioenergetics as well as other essential aspects of cellular physiology. The mitochondrial processes that are essential for the structural and functional integrity of skeletal muscle, and how these processes are regulated in health and disease are poorly defined. It is becoming increasingly clear that fundamental mechanisms underlying the development, function, and maintenance of skeletal muscle are conserved across metazoans. Thus genetic model organisms are poised to make significant contributions to our understanding of these mechanisms. In our previous studies, we have used Drosophila as a model to demonstrate the importance of Numb/Notch signaling and asymmetric progenitor cell division during muscle development, and PINK1/Parkin-directed mitochondrial quality control in skeletal muscle maintenance. We have also used the fly neuromuscular junction (NMJ) as a model to dissect synaptic mechanisms involved in age- related neurodegenerative diseases. In our most recent studies, we have found that protein quality control in the mitochondrial intermembrane space (IMS) is important for skeletal muscle function and maintenance. We found that dipeptide repeats (DPRs) derived from unconventional translation of the GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72, the most common genetic cause of amyotrophic lateral sclerosis (ALS) called c9ALS, disrupt mitochondrial function by altering IMS proteostasis and inner membrane (IM) architecture. Our genetic modifier screens identified a number of signaling pathways in mitigating this ALS-related muscle pathology. The goal of this proposal is to use proteomic, molecular genetic, and cell biological tools to define the mechanism of action of the identified genetic pathways, in an effort to achieve a holistic view of the regulation and function of mitochondrial IM architecture in skeletal muscle function and maintenance. Two Specific Aims will help us reach this goal. In Aim 1, we will examine the molecular mechanisms of how c9ALS disease gene product disrupts muscle mitochondrial IMS/IM structure and function. Novel genetic tools will be used to perform ultrastructural studies and find the interactome of DPR within these structures. In Aim 2, we will delineate the cellular quality control mechanisms that maintain IMS/IM integrity by restraining the synthesis or promoting the metabolism of DPR. The role of these quality control mechanisms in maintaining mitochondrial and skeletal muscle structure and function during normal aging will also be examined. These studies will significantly advance our understanding of the role of mitochondria in maintaining skeletal muscle structure and function. Results from this study promise to inform the development of novel and rational muscle-targeting medicine for ALS and conditions such as sarcopenia.

Alzheimer's disease (AD) remains a looming public health crisis, despite intensive research and pharmaceutical development efforts. No effective treatment option is currently available that can halt the disease process. The recent failures of high-profile clinical trials targeting the amyloid plaques and neurofibrillary tangles, the pathological hallmarks of the AD identified by Dr. Alois Alzheimer more than a century ago and the focus of extensive research and pharmaceutical development efforts, suggest that new directions in delineating the pathogenic mechanisms of AD are warranted before effective treatment of the disease can be achieved. Mitochondria are dynamic and complex organelles with essential roles in many aspects of biology, from energy production and intermediary metabolism to intracellular signaling and apoptosis. These broad functions position mitochondrion as a central player in human health. In neurons, mitochondria and synapses are intimately linked. In addition to the central role of mitochondria in bioenergetics, they are also critically important for maintaining cellular Ca2+ homeostasis. Ca2+ uptake by mitochondria helps buffer cytosolic Ca2+ transients arising from neuronal activation, protecting against the detrimental effects of bursts of Ca2+ influx. Under basal conditions, Ca2+ entry into mitochondria is needed for normal neuronal physiology. The ER- mitochondria contact site (ERMCS) are recognized as key cellular structures regulating mito-Ca2+ homeostasis. Moreover, there is an emerging recognition of ERMCS impairment in neurodegenerative diseases including AD. How ERMCS and mito-Ca2+ homeostasis are altered, and their contribution to disease phenotypes in in vivo settings, however, are not well understood. The goal of this proposal is to test the central hypothesis that an interplay between APP metabolism and ERMCS directs ER-mitochondrial Ca2+ signaling, and that defects in this process contributes to the etiology of AD. To test this hypothesis, we propose to achieve the following Specific Aims in this exploratory project: Aim 1. Examine defects in ERMCS formation in a Drosophila AD model and AD patient derived cells; Aim 2. Test the roles of ERMCS proteins that direct mito-Ca2+ homeostasis in mediating APP function in disease pathogenesis. By providing evidence for the involvement of ERMCS and mito-Ca2+ in APP function at the organellar, synaptic, and organismal levels, these studies will lay the foundation for future studies addressing the regulation and function of ERMCS in normal brain physiology, which will significantly advance our understanding of the fundamental roles of mitochondria and Ca2+ signaling in AD and ultimately offer novel therapeutic strategies.

Project Summary Short stature, hearing loss, retinitis pigmentosa, and distinctive facies (SHRF) is a rare autosomal recessive disorder characterized by short stature, brachydactyly, dysmorphic facial features, hearing loss, and visual impairment. Patients also exhibit mild intellectual disability. SHRF is caused by homozygous or compound heterozygous mutation in the EXOSC2 gene, which encodes a subunit of the RNA exosome complex, a conserved multi-subunit ribonucleolytic complex that controls the 3´ to 5´ processing and degradation of various RNAs in all eukaryotic cells. Nine subunits (EXOSC1-9) form a catalytically inert core that serves as a scaffold for two ribonuclease subunits (EXOSC10 and Dis3). EXOSC2 and EXOSC3 are S1 and KH domain containing RNA-binding proteins that form the exosome cap structure. Intriguingly, despite the sequence similarity and similar positions in the exosome structure occupied by EXOSC2 and EXOSC3, mutations in these subunits result in distinct diseases, with mutations in EXOSC2 causing SHRF and mutations in EXOSC3 causing Pontocerebellar Hypoplasia type 1b (PCH1b), a rare autosomal recessive neonatal/fetal neurodegenerative disease characterized by hypoplasia and atrophy of the cerebellar cortex, dentate nuclei, pontine nuclei and inferior olives. That mutation in core subunits of a seemingly universally required RNA exosome complex can result in distinct diseases reflects inherent complexity in the organization, function, and regulation of this fundamental machinery of post-transcriptional gene regulation. But our understanding of the mechanistic basis underlying these processes is very limited. We hypothesize that RNA exosome subunits are assembled into different subcomplexes with different RNA substrate engagements, and that these subcomplexes may function in a tissue or cell type-specific manner. To test this hypothesis, we propose to employ the recently developed proximity labeling using the engineered enzyme ascorbate peroxidase 2 (APEX2) to systematically identify proteins and RNAs in the immediate proximity of EXOSC2 in mammalian cell culture models, including iPSC-derived neuronal and muscle models, and in vivo Drosophila models. The functional involvement of newly identified factors in exosome biology and SHRF pathogenesis will be tested in Drosophila models. Successful execution of this project will not only lead to new knowledge on the composition, regulation, and tissue-specific requirement of the RNA exosome complex, but also shed light on the pathogenesis of RNA exosome-linked diseases, from SHRF, PCH, SMA and pulmonary fibrosis to cancer, diseases affecting multiple body systems. It is therefore expected that findings from this study will be applicable to the missions of multiple NIH Institutes or Centers (ICs), one of the stated Research Objectives of this R21 funding opportunity.