Eric J. Huang, MD PhD - US grants

[unreadable] DESCRIPTION (provided by applicant): Apoptosis plays important roles in the developing nervous system and in the maintenance of homeostasis in adult brains. Aberrations in the regulation of apoptosis contribute to pathogenesis of neurodegenerative diseases, including hereditary sensory and autonomic neuropathy, ALS, Huntington's Disease, Parkinson's Disease, and Alzheimer's Disease. One major mechanism to control cell death is by transcriptional regulation of members in the Bcl-2 family. For instance, p53 induces neuronal apoptosis by up-regulating Bax and inhibiting Bcl-xL expression. In contrast, homeodomain transcription factor Brn3a antagonizes p53 and promotes the expression of Bcl-2 and Bcl-xL. While these results suggest that p53 and Brn3a may control the delicate balance of neuronal death and survival, it remains unclear if these two molecules interact directly or if they are regulated through a common signaling mechanism. Our data favor the latter model and indicate that homeodomain interacting protein kinase 2 (HIPK2) induces neuronal apoptosis by exerting opposing effects on p53 and Brn3a. Expression of HIPK2 in sensory neurons activates apoptosis through up-regulation of p53 target gene Bax and suppression of Brn3a target Bcl-xL. Consistent with these data, HIPK2-induced apoptosis can be partially inhibited by Brn3a, Bcl-xL and Bcl-w. Furthermore, the effect of HIPK2 is much attenuated in p53 -/- neurons and is completely abolished in BaX/neurons. HIPK2 is abundantly present in the nervous system and the subcellular localization of HIPK2 in sensory neurons appears to coincide with the stages when neurons receive neurotrophins from the target tissue, suggesting that the activity of HIPK2 may be regulated through neurotrophin signaling pathways. These results lead us to hypothesize that HIPK2 is a key component that regulates the neuronal death during development and in stress-induced pathological conditions. To test this hypothesis further, we propose to investigate the upstream signaling pathways that regulate HIPK2 activity. In particular, we will focus on the roles of JNK pathway in HIPK2 functions (Aim 1). To determine the in vivo functions of HIPK2, we have deleted the HIPK2 gene and will investigate the neurological phenotype of HIPK2 / mutants in developmentally programmed cell death and in injury-induced apoptosis paradigms (Aim 2). Finally, we will study the detailed mechanisms of HIPK2-induced, p53-dependent regulation of Bax and related pro-apoptotic genes. We will also determine if HIPK2-mediated activation of additional targets, such as p53-related molecule p73, contributes to neuronal apoptosis. Our long-term goal is to elucidate HIPK2-induced signaling pathways in neuronal apoptosis. Discoveries from this project will contribute to future design of HIPK2 inhibitors that can promote neuronal survival in neurodegenerative diseases [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application for an NIH RC1 Challenge Grant in Health and Science Research addresses the following Challenge Areas: (04) Clinical Research and (15) Translation Science. The specific Challenge Topics it addresses are 04-DK-104, Improve the diagnosis, staging and treatment of diseases of the liver;15-CA-111, Infectious Disease and Inflammation in Cancer, and 04-DK-111, Pilot and feasibility clinical research studies in diabetes, obesity, and metabolic, endocrine, digestive, liver, renal and urological diseases. Hepatitis B virus (HBV) is a major cause of serious liver diseases, including chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) throughout the world and the US, especially among minorities such as African Americans, Native Americans, and Asian Americans. HCC has a dismal prognosis unless detected early, but current screening methods have low sensitivity and specificity and are expensive. Recent clinical data have pointed to an association between HCC and HBV mutants with deletions in the preS2 region of the surface gene. We hypothesize that preS2 mutant HBV genomes play a causal role in HBV-associated carcinogenesis, and hence the appearance of these mutants in patients can be used as a marker for high risk of HCC. We will test this hypothesis, by confirming an association between preS2 mutants and advanced stages of hepatitis B with a cross-sectional study, and determining if the presence of these mutants can be used as a marker for increased risk of HCC development in patients with chronic hepatitis B in a case-control study. These studies, unlike previous ones, will be performed on samples from American patients. It is anticipated that these experiments will provide direct evidence on the possible utility of preS2 mutants as a marker of HCC risk in chronic hepatitis B, as well as lead in the future to the identification of molecular targets for the prevention and/or therapy of HCC. PUBLIC HEALTH RELEVANCE: Hepatitis B virus is a major cause of suffering and death in the world, by causing liver injury, cirrhosis (liver scarring) and, most importantly, liver cancer. Current treatment for liver cancer is inadequate, to a large extent because of our inability to detect the cancer early enough. We hope that our research can lead to the development of new ways for the early detection and hence treatment of liver cancer in these patients.

DESCRIPTION (provided by applicant): This R21 application is prepared in response to PA-10-138, Development of Animal Models and Related Biological Materials For Research. In this application, we propose to establish both cellular and animal models that will allow us to characterize the mechanism of FUS/TLS mutations. The FUS/TLS locus on human chromosome 16 is originally identified to encode an oncogene implicated in malignant liposarcoma and myeloid leukemia. More recent evidence shows that mutations in FUS/TLS are found in familial cases of amyotrophic lateral sclerosis (ALS). These results indicate that further investigations to the mechanisms of FUS/TLS gene will have profound impacts on many disciplines in biomedical research, including cancer biology, neurodegeneration, aging, and mental health. Although most mutations are found in the highly conserved C-terminal region of the FUS protein, the precise pathogenic mechanisms for these mutations have not been fully elucidated. Among the FUS mutations, the most common form in adult ALS patients is the R521C mutation. In addition, our recent study indicates that the P525L mutation in FUS leads to a more aggressive and rapidly progressive form of ALS in young patients. Currently, it is unclear how FUS-R521C and FUS-P525L cause neuronal degeneration, or why FUS-P525L tends to affect younger patients. Our recent data, however, indicated that the spinal motor neurons in patients with P525L mutation show mislocalization of mutant FUS proteins in neuronal cytoplasm with abnormal FUS protein aggregates that are associated with markedly disorganized intracellular organelles, including endoplasmic reticulum (ER) and mitochondria. These results lead us to hypothesize that mutant FUS proteins dominantly inhibit normal RNA and protein synthesis and thereby suppressing neuronal functions and survival. Our objectives are to further investigate the mechanisms of mutant FUS proteins in neuronal degeneration using both primary neurons and transgenic mice. Since synaptic degeneration plays a central role in neurodegenerative diseases, we believe that the establishment of these models will contribute to understanding of the mutant FUS proteins in the development and maintenance of synaptic connectivity of motor neurons within the spinal cord and at the neuromuscular junction (NMJ). We propose two specific aims to achieve these goals. In Aim 1, we will characterize the mechanisms by which FUS-R521C and FUS-P525L mutant proteins affect synaptic degeneration and cell death in primary neurons. In Aim 2, we propose to characterize if mice expressing FUS-R521C or FUS-P525L develop motor behavioral phenotype and motor neuron pathology similar to patients with corresponding mutations. We believe that the establishment of these models will have important contributions to the missions of many institutions at NIH, and fulfills the criteria of high rewards and high impacts for the R21 funding mechanism. Once available, these models will serve as novel platforms to identify new therapeutic targets for diseases caused by FUS/TLS mutations.

Project Summary (Core B) The primary goal of the Neuropathology Core (Core B) is to provide high quality human fetal, neonatal and pediatric brain tissues for all the components of the program project. In addition, Core B will also collaborate with researchers and project leaders in this PPG to characterize cell lineages in the developing human brains and develop state-of-the-art molecular markers that will facilitate the discovery and characterizations of neuronal and glial lineages during normal human brain development and in hypoxic ischemic injury conditions. Core B will provide full-time histopathology services that facilitate the progress among the research projects in this PPG as well as coordinate activities with the internal and external advisory committees. In essence, the aim of the Neuropathology Core is to function as a centralized facility where human brain tissues and advances in histopathology techniques can be utilized to provide support and integration of services for all investigators. The core director will work with the project leaders and make decisions regarding the use of core services. As an active member of the PPG, the core director will participate in weekly meetings on Fridays regarding administrative and scientific matters such as research directions, requests for specific human tissues, data analyses, collaborations and presentations of data. The core director, in consultation with Dr. Rowitch, Dr. Alveraz-Buylla and Dr. Kriegstein, will evaluate the specific needs and cost-effectiveness in order to maximize the service of Core B to each research project. The core director will ensure that quality control is provided at the highest level. Additional quality control will be achieved through the use of our internal and external advisory committees.

PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease, is an adult-onset neurodegenerative disease that affects upper and lower motor neurons. The key clinical features in ALS patients include muscle wasting, and progressive loss of spinal motor neurons and upper motor neurons and their axons in the lateral columns of the spinal cord. The past 10 years have witnessed a tremendous expansion in the molecular mechanisms of this devastating disease thanks to the discoveries of genetic mutations that are causally linked to both familial ALS (FALS) and sporadic ALS (SALS). Characterizations of these ?ALS disease genes? suggest that dysfunctions in protein homeostasis via the ubiquitin-proteasome pathways (proteostasis) might contribute to the pathogenesis and disease progression in ALS. Consistent with the genetic data, a key pathological feature in FALS and SALS is accumulation of misfolded proteins in motor neurons, which disrupts normal neuronal functions, including axonal transport, mitochondrial bioenergetics, gene expression, and synaptic connectivity. Persistent accumulation of misfolded proteins eventually triggers endoplasmic reticulum (ER) stress-induced cell death, which leads to neurodegeneration through mechanisms that are poorly understood. This proposal focuses on the neuronal cell death mechanism downstream of the IRE1? pathway of ER stress. We show that ER stress, induced pharmacologically or by mutant SOD1 proteins, activates a highly conserved kinase HIPK2 (homeodomain interacting protein kinase 2) to promote neuronal cell death. Biochemical evidence shows that HIPK2 acts downstream of IRE1?-ASK1 and upstream of JNK to promote ER stress-mediated cell death. In addition, proteomics, phospho-peptide mapping and mutagenesis further show that ER stress activates HIPK2 by promoting phosphorylation on specific Serine and Threonine residues within the kinase domain. Using phospho-HIPK2-specific antibodies, we show that HIPK2 activation in the spinal cord precedes symptom onset in SOD1G93A mice. Importantly, loss of HIPK2 in SOD1G93A;Hipk2-/- mice mitigates neurodegeneration, delays disease onset and prolongs survival. Finally, we have extended our findings of HIPK2 in ER stress to human disease using a large number of spinal cord tissues from FALS and SALS patients. Together, these results support the hypothesis that HIPK2 is an essential target in the downstream of IRE1? pathway that promotes ER stress-induced neuronal cell death in ALS. We propose three multidisciplinary Aims to investigate the robust, yet previously unappreciated role of HIPK2 in ER stress-induced cell death mechanism in ALS. Results from these studies will not only address a major challenge in understanding disease mechanism in ALS, they will also provide new directions to develop potential therapeutic targets to mitigate neuronal cell death in ALS.

PROJECT SUMMARY Alzheimer's Disease (AD) is characterized by the progressive accumulation of abnormally cleaved A? amyloid peptides and hyperphosphorylated tau proteins, which lead to amyloid plaques and neurofibrillary tangles, respectively. While it remains unclear what triggers these proteinopathies, several lines of evidence indicate that defects in intracellular trafficking may regulate AD pathogenesis. Importantly, emerging evidence suggests that AD-related proteins, including tau, amyloid precursor protein (APP) and A? amyloid peptides are secreted via exosomes. Despite these findings, it remains unclear what regulates the formation and packaging of exosomes, whether exosome biogenesis is functionally connected to intracellular trafficking of disease-related proteins, whether neurons and glia develop different mechanism to process these proteins, and if so, how abnormal proteostasis in neurons and glia cooperatively promotes neurodegeneration. We have discovered a new pathway in which the autophagy machinery specifies packaging and secretion of proteins within exosomes. Traditionally studied as an autodigestive pathway that promotes cell survival during stress, autophagy also promotes the unconventional secretion of proteins lacking N-terminal signal sequences. Using a proximity-based biotinylation (BioID) proteomics strategy, we have uncovered ~90 novel putative targets of autophagy-dependent secretion, including numerous proteins released within exosomes. These proteins biochemically interact with MAP1LC3B, a mammalian ATG8 isoform and autophagy regulator crucial for cargo sequestration. Based on these results, we hypothesize that the autophagy machinery mediates the LC3-dependent recruitment and packaging of specific intracellular cargo for their secretion via exosomes. Furthermore, we hypothesize that autophagy controls a delicate balance of secretion and intracellular trafficking of disease-relevant proteins in neurons and glia to promote neurodegeneration in AD and FTD. To test these predictions, we will: 1) Dissect whether and how autophagy specifies exosome packaging and secretion in normal and Alzheimer neural cell populations; and 2) Delineate how lysosomal dysfunction in AD and FTD impacts LC3-dependent exosome packaging and proteostasis. These studies are uniquely poised to define new functions for the autophagy machinery in the biogenesis and secretion of exosomes and to delineate its contributions to AD pathogenesis. This multi-PI R01 application synergistically merges the unique expertise of Dr. Jayanta Debnath in the cell biology of autophagy and Dr. Eric Huang in the molecular mechanisms of neurodegenerative diseases to address the goals of RFA-AG-17-051 by uncovering new machineries directing exosome biogenesis and the secretion of exosomal cargo molecules in AD.

Project Summary (Core B) In the previous funding cycle, the Neuropathology Core (Core B) established a highly effective brain tissue banking system that greatly facilitates the success of research projects in this PPG. In the renewal proposal, Core B will continue its mission to provide high quality 2nd ? 3rd trimester, perinatal and early postnatal human brain tissues from the Autopsy Service in the Department of Pathology at UCSF for all the components of the PPG. In particular, Core B will focus on preparing tissues for single cell RNA-seq (scRNA-seq), single nucleus RNA-seq (snRNA-seq), RNAscope and ultrastructural analyses, which will prepare researchers and project leaders in this PPG to characterize the molecular and cellular mechanisms of cell lineages in the developing human brains. Core B will develop state-of-the-art molecular markers that will facilitate the discovery and characterizations of neuron-glia interactions during normal human brain development. Core B will provide full- time histopathology services that facilitate the progress among the research projects in this PPG as well as coordinate activities with the internal and external advisory committees. In essence, the aim of the Neuropathology Core is to function as a centralized facility where human brain tissues and advances in histopathology techniques can be utilized to provide support and integration of services for all investigators. The core director will work with the project leaders and make decisions regarding the use of core services. As an active member of the PPG, the core director will participate in weekly meetings regarding administrative and scientific matters such as research directions, requests for specific human tissues, data analyses, collaborations and presentations of data. The core director and co-director, in consultation with Dr. Alveraz-Buylla, Dr. Rowitch, Dr. Fancy, and Dr. Piao, will evaluate the specific needs and cost-effectiveness in order to maximize the service of Core B to each research project. The core director will ensure that quality control is provided at the highest level. Additional quality control will be achieved through the use of our internal and external advisory committees.

Project Summary Frontotemporal dementia (FTD) is an early onset neurodegenerative disease, and the second most common cause of dementia in patients 60 years or younger. The majority of familial FTD are caused by intronic hexanucleotide (CCCCGG) repeat expansion in chromosome 9 open reading frame 72 (C9orf72) gene and by dominant mutations in the Progranulin (GRN) gene, which account for 25% and 15% of familial FTD cases, respectively. These mutations cause haploinsufficiency in both genes and lead to abnormal protein aggregation involving RNA binding protein TDP-43 in neuronal nuclei and cytoplasm. The goal of the parent grant (R01 AA027074-03) is to test the hypothesis that loss of PGRN disrupts neuroimmune interaction in the thalamo- cortical circuit in Grn-/- mice. The purpose of this Diversity Supplement is to expand the scope of the parent grant and characterize the potential interaction of C9orf72 and progranulin in neurodegeneration. To this end, the proposed trainee, Naznin Jahan ? a 4th year graduate student in the BMS Graduate Program at UCSF, has established an aging cohort of C9orf72-/-, Grn-/-, and C9orf72-/-;Grn-/- double KO (DKO) mice. The trainee?s results showed that C9orf72-/-;Grn-/- DKO mice exhibit age-dependent neuroinflammation and neuronal loss that are more pronounced than those seen in C9orf72-/- and Grn-/- mice. These findings support the intriguing hypothesis that simultaneous loss-of-function (LOF) in C9orf72 and GRN genes synergistically disrupts glial homeostasis and promote neuronal degeneration in an age dependent manner. The scope of this Diversity Supplement includes (1) to determine the transcriptomic changes regulated by C9orf72 and Grn in glia-neuron homeostasis, and (2) to expand the transcriptomic data using in situ hybridization (ISH), immunohistochemistry and Western blots. In addition, this Diversity Supplement includes a well-defined 2-year timeline, a detailed Mentoring Plan, and Individual Career Development Plan (ICDP) that will significantly enhance the candidate?s research capabilities and complete her dissertation work on the genetic interactions that cause the age dependent neurodegeneration in mice. Working within the proposed timeline, this supplement will prepare the candidate for her long-term career goal as an academic scientist in the field of neuroimmune interaction and neurodegeneration.