Fen-Biao Gao - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The nervous system is composed of a vast number of neurons that vary dramatically in size and shape. Neurons are highly polarized cells with distinct subcellular compartments, including one or more dendritic processes arising from the cell body and a single, extended axon. Elucidating the mechanisms that control neuronal polarity and dendritic development is of critical importance for understanding the development and plasticity of a functional nervous system. In addition, alternations in the number of dendritic branches and dendritic spines are often found in patients with neurological disorders, such as fragile X syndrome. Fragile X syndrome is the most common form of inherited mental retardation in humans, with an estimated incidence of 1 in 4000 males and 1 in 8000 females. The disorder is caused by the loss of the fragile X mental retardation 1 (fmr1) gene activity. FMR1 is an RNA-binding protein that contains two ribonucleoprotein K homology domains (KH domains) and an arginine- and glycine-rich domain (RGG box). The physiological function of FMR1 in neural development remains largely unknown. The long-term goal of this laboratory is to understand the molecular mechanisms underlying dendritic outgrowth, branching, and remodeling during development. The peripheral nervous system (PNS) of the fruitfly Drosophila is an ideal model system for these studies. PNS neurons can be individually identified, and their dendritic morphology can be studied in real time in living animals. A large number of genes identified in PNS also affect dendritic development of central nervous system (CNS) neurons. In addition, the Drosophila model allows powerful genetic and molecular manipulations. Recently, we have generated specific mutations in the Drosophila fmr1 (dfmr1) gene. Our preliminary studies indicate that dfmr1 mutations primarily affect the formation of higher-order dendritic branches. In this proposal, we will carry out a series of experiments to further understand how FMR1 controls dendritic development. Specially, (1) we will further characterize the dendritic overextension phenotype caused by dfmr1 mutations, (2) we will investigate how dFMR1 functions at the mechanistic level, and (3) we will use genetic approaches to identify other proteins that also control dendritic development and may interact with dFMR1. Molecular mechanisms underlying many biological processes are highly conserved throughout evolution. Studies of the mechanisms that control dendritic development in Drosophila may help us understand similar processes in human brains. The insights gained from these studies may also contribute to our understanding of fragile X syndrome. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): ALS is the most common form of motor neuron degenerative disease, affecting adults during mid to late life, occurring in 2-3 per 100,000 people. The disease is characterized by progressive degeneration of upper and lower motor neurons in the brain and spinal cord that leads to muscle weakness and atrophy. In majority of cases ALS is not associated with any known mutations, and the etiology is still poorly understood. It has long been suspected that environmental toxicants play a role in the etiology of ALS. However, how environmental toxicants, such as heavy metals, affect normal development and function of neurons and how they contribute to pathology of ALS and other neurodegenerative disorders are still poorly understood, especially at the molecular level. [unreadable] We propose to use a new assay system in Drosophila to study the effects of chronic exposure to environmental toxicants on the structural integrity of neurons in vivo. Unbiased genetic screens will be carried out to identify molecular players whose mutations can either enhance or suppress heavy metal neurotoxicity. These studies represent an innovative approach to examine the effects of environment-gene interactions on neuronal structures, which will likely make significant contributions to our understanding of the molecular mechanisms through which genetic alternations and environmental insults work together to lead to pathological changes involved in neurodegenerative diseases, such as ALS. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Angelman syndrome is a severe developmental brain disorder characterized by mental retardation, seizures, abnormal gait, hyperactivity, frequent laughter, and other abnormalities. Several lines of evidence strongly implicate the loss of the maternal human Ube3A (hUbe3A) gene in most, if not all, cases of Angelman syndrome. Ube3A encodes the E6-AP ubiqutin E3 ligase whose substrates remain largely unknown. How the loss of UbeSA activity leads to clinical symptoms of Angelman syndrome patients needs further investigation. In the Drosophila genome, the gene CG6190 encodes the Drosophila Ube3A (dUbeSA), a protein highly homologous to hUbeSA at the amino acid level. Therefore, Drosophila offers an excellent model system to dissect the molecular and cellular functions of Ube3A. Here we propose to test the hypothesis that UbeSA is required for the proper formation of neuronal fine structures and for the normal development of neuronal connectivity. The defects in these neurodevelopmental processes may contribute to the pathogenesis of Angelman syndrome. We will generate dUbeSA loss-of-function mutant flies and characterize dUbeSA function in neuronal development. We will also use these mutant flies to identify some evolutionary conserved key substrates of UbeSA ubiquitin ligase that mediate the effects of UbeSA on the brain development and function. These studies will provide important insights into the molecular and cellular mechanisms underlying this devastating developmental brain disorder and into the potential avenues for therapeutic interventions. Gladstone Institute of Neurological Disease, The J. David Gladstone Institutes, San Francisco, CA PHS 398 (Rev. 09/04) Page 2 Form Page 2 3 Principal Investigator/Program Director (Last, First, Middle): Gao, Fen-BiaO KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Fen-Biao Gao, Ph.D. fenbiao The j. David Gladstone institutes Principal Investigator Van Li, Ph.D. N/A The J. David Gladstone Institutes Postdoctoral Fellow Fay Wang, Ph.D. N/A The J. David Gladstone Institutes Postdoctoral Fellow OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Human Embryonic Stem Cells [x] No D Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://stemcells.nih.gov/reqistry/index.asp. Use continuation pages as needed. If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used. Cell Line Disclosure Permission Statement. Applicable to SBIR/STTR Only. See SBIR/STTR instructions.Yes DNO PHS 398 (Rev. 09/04) Page 3 Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. 4 Principal Investigator/Program Director (Last, First, Middle): GaO, Fen-BiaO The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page J Description, [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Defects in the endosomal-lysosomal pathway have been implicated in several neurodegenerative diseases but the detailed underlying molecular mechanisms remain largely unknown. Frontotemporal lobar degeneration (FTLD) is a progressive neurodegenerative condition associated with focal atrophy of the frontal and/or temporal lobes. FTLD is one of the most common forms of presenile dementia. Increasing clinical and molecular evidence indicates that FTLD and amyotrophic lateral sclerosis share many common pathogenic mechanisms. Indeed, several genes, including CHMP2B, VCP, TDP-43, FUS, Ubiquilin 2, and C9ORF72, have been implicated in the molecular pathogenesis of both diseases. During the first funding cycle of this R01 grant, we established a neuronal cell model and a Drosophila model of mutant CHMP2B toxicity and investigated the roles of ESCRTs and autophagy in neurodegeneration. To more closely model human disease, we established a novel transgenic mouse model that exhibits several key features of FTLD-associated neurodegeneration. In this renewal application, we propose to carry out molecular, cellular, genetic, and behavioral analyses to further characterize this novel mouse model of FTLD, with the goal of gaining mechanistic insights into pathogenic events in vivo. The proposed studies will significantly enhance our understanding of disease mechanisms in FTLD and may reveal novel targets for therapeutic interventions.

DESCRIPTION (provided by applicant): An early step in neurogenesis is the generation of neuronal precursor cells from the naive neuroepithelium. Although transcription factors and several signaling pathways that promote early neurogenesis have been identified, the mechanisms that ensure the generation of precise numbers of neuronal precursors from the neuroepithelial cells remain to be further defined. One of the best model systems for understanding early neurogenesis is sensory organ precursor (SOP) formation in the Drosophila peripheral nervous system. A small number of cells, known as the proneural cluster, express proneural genes that encode basic helix loop helix (bHLH) proteins and become competent to develop into SOPs. The SOP emerges from the proneural cluster through the actions of "neurogenic" genes (e.g., Notch and Delta) that maintain proneural gene expression at high levels in the SOP and at low levels in adjacent epithelial cells. The ligand Delta binds to the Notch receptor, which in turn regulates the differential expression of proneural genes through the actions of Suppressor of hairless [Su(H)] and the Enhancer of split complex. Senseless (Sens), a nuclear protein with four zinc fingers that is required to upregulate and maintain proneural gene expression in SOPs, is expressed at a high level in SOPs. In adjacent epithelial cells in the proneural cluster, Sens is expressed at a low level and suppresses proneural gene expression. These differences in the expression and function of Sens are essential for proper SOP formation. The mechanism of differential regulation of these genes required for the accurate production of neuronal precursor cells is unclear. Recently, we generated microRNA-9a loss-of- function mutant flies and found that microRNA-9a normally inhibits neuronal fate in non-SOP cells by downregulating Sens expression. In this application, we propose to further dissect the feedback loops between proneural genes and microRNA-9a. Using genetic screens, we will also identify other key targets of microRNA- 9a, including potential novel players important for early neurogenesis. Since many microRNAs are 100% conserved at the nucleotide level from flies to mammals, our findings will have important implications for mammalian neurogenesis as well and may provide novel insights into neurodevelopmental disorders.

DESCRIPTION (provided by applicant): TDP-43 is a major pathological protein in amyotrophic lateral sclerosis (ALS) and some forms of frontotemporal dementia (FTD) and is also present in ubiquitinated inclusions seen in other neurodegenerative diseases. How TDP-43 contributes to age-dependent neurodegeneration is largely unknown. TDP-43, an evolutionarily highly conserved RNA-binding protein mostly localized to the nucleus, participates in transcriptional repression, alternative splicing, mRNA trafficking, and possibly other aspects of RNA metabolism. However, its functions in postmitotic neurons have not been extensively studied. Normally, TDP-3 has a diffuse nuclear distribution. In diseased neurons, however, TDP-43 and its processed fragments aggregate into cytoplasmic ubiquitinated inclusions. Thus, loss of the normal function ofTDP-43 might contribute to neurodegeneration, perhaps in concert with other mechanisms, such as a toxic gain-of-function for TDP-43 fragments. In this R21 application, we propose to investigate the normal functions of endogenous TDP-43 in postmitotic neurons, which will likely provide important insight into the molecular mechanisms underlying TDP-43-related neurodegeneration. PUBLIC HEALTH RELEVANCE: In this application, we will test the hypothesis that loss of TDP-43 activity affects neuronal structural integrity therefore contributes to eventual neurodegeneration associated with a number of age-dependent neurodegenerative diseases. To this end, we will use fruitfly Drosophila as our primary model system for all the proposed studies.

DESCRIPTION (provided by applicant): Several mental disorders are closely associated with defects during neuronal development in early life. Abnormalities in neuronal differentiation and synaptogenesis may contribute to fragile X syndrome, Rett syndrome, autism, and other mental disorders. Post-transcriptional regulation by microRNAs (miRNAs) has emerged as an important mechanism for controlling gene expression in animal development; however, the exact functions of miRNAs in neuronal differentiation and function are poorly understood. Moreover, the genes and pathways regulated by miRNAs in the nervous system are largely unknown. miRNAs are 21-23-nt endogenous noncoding RNAs processed from 70-80-nt precursors that are mostly transcribed by RNA polymerase II and form stem-loop structures. miRNAs regulate gene expression by repressing translation or cleaving messenger RNAs. Estimated to comprise 1-5% of animal genes, miRNAs are thought to regulate the expression of a large number of target genes in developmental processes. A few miRNAs are specifically expressed in mammalian brains, suggesting unique regulatory roles in neuronal development and function. Indeed, miRNAs have been implicated in left/right neuronal asymmetry in Caenorhabditis elegans, photoreceptor formation and early neurogenesis in Drosophila, brain morphogenesis and neurogenesis in zebrafish, and neuronal differentiation in mammals. However, loss-of-function approaches have not been widely used to study the roles of miRNAs in the nervous system or other development processes. In Drosophila, for instance, only a few miRNAs have been studied in vivo using loss-of-function approaches, including miR-9a whose function in the specification of sensory organ precursors was reported by our laboratory. In this application, we propose to use in vivo genetic and molecular techniques and genomic approaches to elucidate the role of translational control by miRNAs in neuronal development in Drosophila. We will also investigate the underlying molecular mechanisms and validate and characterize one or two bona fide miRNA targets. The proposed studies will provide important novel insights into the neuronal functions of the miRNA pathway in intact animal models and will further our understanding of human mental disorders. Our findings may also help develop new avenues for therapeutic interventions for these devastating illnesses.

DESCRIPTION (provided by applicant): Frontotemporal lobar degeneration (FTLD) is a progressive neurodegenerative condition associated with focal atrophy of the frontal and/or temporal lobes. Recent exciting progress indicates that FTLD and amyotrophic lateral sclerosis (ALS) share pathogenic mechanisms. For instance, TDP-43, an evolutionarily conserved RNA- binding protein mostly localized to the nucleus, is a major pathogenic protein involved in FTLD and ALS, and in other neurodegenerative diseases. However, little is known about how TDP-43 contributes to age-dependent neurodegeneration. TDP-43 contributes to several aspects of RNA metabolism, and disease initiation or progression may involve both loss of the normal function of TDP-43 and toxic gain-of-function mechanisms. To dissect these complex mechanisms in detail, proper in vivo animal models are critically important. We propose to establish novel Drosophila models of TDP-43 toxicity and investigate how disease mutations compromise the function of TDP-43 and lead to neuronal dysfunction in vivo. These studies will likely provide important insights into the molecular pathogenic mechanisms of several neurodegenerative diseases that involve TDP- 43 pathology. PUBLIC HEALTH RELEVANCE: In this proposal, we will establish a novel fly model of neurodegeneration and perform a number of genetic and molecular experiments. These studies will offer novel mechanistic insights into the neurotoxicity of mutant TDP-43, which will likely enhance our understanding of molecular pathogenic mechanisms of frontotemporal dementia and amyotrophic lateral sclerosis.

DESCRIPTION (provided by applicant): We propose to establish a comprehensive, validated repository of adult human dermal fibroblasts and human induced pluripotent stem cell (hiPSC) lines from frontotemporal dementia (FTD) patients with genetically defined mutations and familial, non-mutation carrying controls. hiPSCs hold tremendous promise for the development of in vitro FTD models for studying disease pathogenesis in relevant human cell types that would otherwise be impossible to obtain, such as human neurons. Using an established, collaborative, multi- institutional approach, we will bank adult human dermal fibroblasts from FTD patients carrying common mutations in the genes currently known to cause FTD: tau (MAPT), C9ORF72, and progranulin (GRN). In Aim 1, we will recruit both FTD patients with defined genetic mutations and control subjects. Comprehensive and longitudinal clinical evaluations will be linked to each cell line, allowing us to correlate disease characteristics with molecular phenotypes. In Aim 2, we will reprogram fibroblasts into hiPSCs by non-DNA-integrating technologies with which we have had recent success. In addition, we will further create EGFP reporter lines for monitoring and standardizing differentiation protocols in FTD-relevant cell types such as forebrain neurons. We will also correct selective mutations to create isogenic control lines so that we can precisely differentiate mutation-specific phenotypes from the noise of inter-individual variability. In Aim 3, we will derive and validate human neurons to model and study FTD pathogenesis in culture and to deliver hiPSC lines with robust phenotypes for FTD research and drug development. Based on our previous research experience in RNA and Tau biology and pathophysiology, we will focus on human neurons with GGGGCC repeat expansions in C9ORF72 and MAPT mutations. All cell lines will be banked at the Coriell Institute and will be accessible to the worldwide FTD research and drug development community. These resources should significantly alter the FTD research landscape by accelerating discovery.

DESCRIPTION (provided by applicant): Frontotemporal dementia (FTD) is a major presenile age-dependent dementia characterized by several clinical features including progressive behavioral changes and language impairments. TDP-43 is a major pathological protein in FTD whose translocation from the nucleus to the cytoplasm in diseased neurons suggests loss of TDP-43 nuclear function may be a key pathogenic mechanism. Several studies including our own implicate defects in the microRNA (miRNA) pathway are one of the important molecular alterations downstream of TDP-43. Using Drosophila as a model, we found that microRNA-92a/b are significantly downregulated in Drosophila TDP-43 (dTDP-43) loss of function mutants. To further understand miRNA functions at the mechanistic level, we generated microRNA-92a, and microRNA-92b single and double mutant fly lines. In this R21 application, we propose molecular, cellular, genetic analyses to further investigate how this nervous system-enriched and evolutionarily conserved but understudied miRNA family functions downstream of TDP-43 to regulate synaptic and dendritic structures in Drosophila and human neurons. These studies will likely provide novel insights into the complex molecular regulatory mechanisms that may contribute to early disease phenotypes in FTD and related neurodegenerative disorders.

? DESCRIPTION (provided by applicant): Frontotemporal dementia (FTD) is a fatal disease associated with focal atrophy of the prefrontal and anterior temporal cortex. FTD is the second most common cause of dementia under age 65 (after Alzheimer's disease), and there is no cure. Compared to other major neurodegenerative disorders, very little is known about its pathogenic basis at molecular, synaptic, and circuit levels. Behavioral abnormalities, characterized by marked changes in personality and social conduct are hallmarks of FTD. As many as 50% of FTD patients have a family history of the disease, suggesting a strong genetic component. Recent molecular genetic studies have identified a number of FTD-causing genes, including CHMP2B, progranulin (GRN), and C9ORF72, paving the way for in-depth investigation of pathogenic mechanisms of the disease. Interestingly, the clinical outcome in FTD patients carrying different mutations is often strikingly similar, suggesting that the same neural circuits are affected. In an effort to model FTD in animals and elucidate underlying mechanisms, we have generated a conditional transgenic mouse strain that expresses an FTD-causing CHMP2BIntron5 in the forebrain. Mutant mice recapitulate several key features of FTD-associated neurodegeneration phenotypes, including social behavioral impairments. Our studies uncover a marked change in AMPA receptor (AMPAR) composition, leading to abnormal insertion of Ca2+-impermeable AMPARs in synapses of the medial prefrontal cortex (PFC). This AMPAR dysregulation appears to be driven by a loss of the brain-enriched noncoding microRNA miR-124. Importantly, similar changes in miR-124 and AMPARs are also observed in the frontal cortex and iPSC-derived cortical neurons from a subset of patients with behavioral variant FTD (bvFTD). This suggests that miR-124 and AMPAR dysregulation are not limited to CHMP2BIntron5 mutation, and are perhaps a more general pathogenic mechanism for FTD with more common mutations. Based on these results, we propose a novel mechanism of FTD pathogenesis: altered synaptic AMPAR assembly and function in PFC circuits underline the social behavioral impairments in FTD. The goal of this application is to test and establish this AMPAR hypothesis of FTD. We will examine key AMPAR mechanisms and map affected circuits in the medial PFC in an existing (GRN haploinsufficiency) and two newly generated (CHMP2BIntron5 and C9ORF72 AAV transgenic mice with G4C2 repeat expansions) mouse models of FTD (Aim 1), establish the miR-124-AMPAR pathogenic axis and by manipulating this axis, especially CP-AMPARs, restore sociability deficits in mouse models in vivo (Aim 2), and finally, validate the AMPAR hypothesis and explore therapeutic strategies in human patient iPSC-derived cortical neurons harboring different genetic mutations (Aim 3). We will use a multidisciplinary approach combining in vivo gene manipulations, electrophysiology, mouse behavior, and human iPSC technologies. Our results will set the stage for translational studies aimed at developing an AMPAR-based therapeutic strategy for FTD.

Dementia is one of the greatest global health challenges we face in the 21st century. Frontotemporal dementia (FTD) is the second most common form of dementia among people under the age of 60 and its pathogenic mechanisms are poorly understood. FTD and amyotrophic lateral sclerosis (ALS), a predominantly motor neuron disease, share many clinical, pathological, and genetic features. The rapid identification of an array of genes that cause FTD/ALS has opened exciting opportunities to dissect shared pathogenic molecular pathways that may be effective targets for therapeutic intervention. In this appalication, we study the most common genetic cause of FTD/ALS, a GGGGCC repeat expansion in the C9ORF72 gene. This genetic mutation may cause disease through multiple mechanisms, including neurotoxicity induced by dipeptide (DPR) proteins generated through repeat-associated non-AUG (RAN) translation. To study these diverse pathogenic mechanisms, we take advantage of the power of Drosophila genetics including genetic suppressors and enhancers of different FTD/ALS disease genes. Such studies often reveal totally unexpected molecular pathways that shed light on pathogenic mechanisms and suggest novel therapeutic targets. In addition, cortical neurons differentiated from patient-specific induced pluripotent stem cells (iPSCs) and human patient brain tissues will be used as a complementary approach. This integrated approach?combining genetic, cellular, molecular, electrophysiological, and bioinformatics analyses?will enable us to make significant contributions to dementia research in the years to come.

Amyotrophic lateral sclerosis (ALS) is a fatal disease caused by motor neuron degeneration and resulting muscle wasting. The ~6,000 new cases of ALS in the US each year afflicts those >40 years of age. There is no effective treatment for ALS and those who contract the disease die 2-5 years after diagnosis. Most causes of ALS are unknown, but the most common known genetic cause is an aberrant C9ORF72 gene. Within this gene, a DNA GGGGCC hexanucleotide sequence is reiterated perhaps thousands of times, which causes the disease. This G4C2 expansion is transcribed into RNA that in turn generate dipeptide repeat proteins (DPRs), whose toxicity is thought to promote motor neuron degeneration. We have identified regions in C9ORF72 RNA that are the start sites for DPR synthesis. These start (ribosome initiation) sites can be occluded by binding to complementary DNA antisense oligonucleotides (AS-ODNs), which are modified to readily enter cells of the nervous system and stably and specifically bind their target sequences in C9ORF72 RNA. Such AS-ODNs would inhibit DPR production, thereby slowing or inhibiting disease progression. We propose to use human disease neurons in culture and C9ORF72 model mice to test the efficacy and specificity of AS-ODNs to inhibit DPR synthesis and mitigate ALS pathophysiology.  

ABSTRACT Frontotemporal dementia (FTD) is a progressive neurodegenerative disease associated with focal atrophy of the prefrontal and/or temporal lobes. FTD is the second most common form of dementia among people under the age of 65. Many FTD-causing genes have been identified during the last decade, including CHMP2B, GRN, C9ORF72, and TBK1. Some of these genes are also implicated in the motor neuron disease amyotrophic lateral sclerosis (ALS), paving the way for in-depth mechanistic investigation of pathogenic processes in both disorders. In order to reveal common pathogenic mechanisms in different forms of FTD, it is critically important to investigate both common and rare genetic mutations. To this end, in this application, we will focus on the effects of FTD-causing mutations in CHMP2B and TBK1 on the functions of the endosomal- lysosomal and autophagy pathways, two closely linked cellular pathways for degradation of transmembrane and intracellular cargos. We will take advantage of strengths of different experimental systems including fruitfly Drosophila, mouse models of FTD and cortical neurons differentiated from CRISPR-engineered induced pluripotent stem cells (iPSCs). This multidisciplinary approach will greatly enhance our understanding of pathogenic mechanisms of FTD and reveal novel targets for therapeutic intervention.