Li-Huei Tsai - US grants

DESCRIPTION (provided by applicant): The mammalian cerebral cortex is an orderly laminated structure containing six distinct layers of neurons. This structure is constructed during cortical development involving programmed cell migration and positioning. Disruption of brain cytoarchitecture can result in severe consequences including epilepsy, mental retardation and early lethality. A small protein ser/thr kinase Cdk5 in conjunction with its regulatory partners, p35 and p39, plays an indispensable role in these processes. We and others showed that in the absence of Cdk5, neuronal migration is impaired resulting in cell positioning defects in the cerebral cortex, hippocampus, cerebellum and other hind brain structures. We have evidence to suggest that Cdk5 regulates actin and microtubule dynamics as well as cadherin mediated cell adhesion. These functions of Cdk5 may contribute to its role in neuronal positioning. Recently, we have obtained experimental evidence to suggest interactions of cdk5 with other known pathways regulating neuronal migration. Haplo-insufficiency of Lis 1 is responsible for type I lissencephaly, a disastrous neurological disorder in humans featuring abnormal neuronal positioning. We and others showed that mammalian Lis 1 interacts with cytoplasmic dynein and modulates dynein activity. Nudel is a novel protein that interacts with both Lis 1 and dynein. Interestingly, Nudel is a physiological substrate of cdk5 as phosphorylation of Nudel is diminished in brain extract lacking cdk5 kinase activity. The integrin receptors are implicated in migration of many different cell types. We found that cdk5 down regulates focal adhesions. Furthermore, the focal adhesion kinase (FAK) is phosphorylated on serine732 by cdk5. Phosphorylation of S732 is developmentally regulated and abolished in the p35/p39 compound mutant brain extract indicating that FAK is an endogenous substrate of cdk5. We hypothesize that phosphorylation of Nudel and FAK by cdk5 plays a regulatory role in neuronal migration and positioning and propose experiments to decipher the biological consequences of these phosphorylation events. Finally, we will make use of an in vitro system established in my laboratory to study and compare cdk5- and Lis1-dependent migration defects in living brain slices. These experiments should bring novel insight into the mechanism by which neuronal migration and positioning is regulated during cortical development.

DESCRIPTION (provided by applicant) The long-term goal of the research supported by the paternal grant is to analyze developmental regulation of neuronal migration. The serine-threonine kinase cdk5, a focus of interest of the parental laboratory, is one of the key genes in this process. Ablation of cdk5, or its regulatory activators, causes disruption of mouse brain structure, thus identification of cdk5 substrates may provide us with molecules with crucial activities during brain development. Cdk5 interacts with several signaling networks involved in neuronal migration. Among several substrates, cdk5 phosphorylates Nudel, a LIS1/dynein interacting protein. Human brain organization is also disrupted in case of neuronal migration disorders, as lissencephaly (i.e. "smooth brain"), which may be the result of mutations in LIS1. The Reiner laboratory research is focused on the functional analysis of genes involved in lissencephaly. The premise that underlies the collaboration between Dr. Tsai's and Dr. Reiner's laboratory is that combining of knowledge and expertise from both laboratories can enhance significantly the research in each individual laboratory. The Reiner laboratory has gained expertise in understanding protein domains through computational and experimental analysis. The Tsai laboratory has integrated and methods of in vivo analysis to neuronal development. The specific objectives of the current grant are: 1. Fine mapping of the interaction between Nudel and LIS1: the Nudel interphase. 2. Fine mapping of the interaction between Nudel and LIS1: the LIS1 interphase. 3. Determination of the in vivo functions of LIS1 phosphorylation mutants that affect its interaction with Nudel, and Nudel mutants with aberrant interactions with LIS1, in migrating neurons in the cortex. The combination of skills from both laboratories will allow elucidating the molecular interphase of NudeI-LIS1 interactions and their importance during brain development. This research will be done primarily in The Weizmann Institute, Israel, in collaboration with Li-Huei Tsai as an extension of NIH grant # ROINS037007. [unreadable]

DESCRIPTION (provided by applicant): This application addresses Broad Challenge Area (15) Translation Science and Specific Challenge Topic: 15-NS-103 Demonstration of "proof-of-concept" for a new therapeutic approach in a neurological disease. Alzheimer's disease (AD) is an irreversible neurological disorder that progressively attenuates the cognitive abilities of those afflicted, ultimately leading to death. AD is the most common of all neurodegenerative disorders, with an estimated 25 million victims worldwide. As life expectancies continue to rise, AD is becoming increasingly common, and it is estimated that the number of those afflicted with AD will increase to 114 million by the year 2050, and cost over $700 billion a year if nothing is done to curb the disease. Despite intensive studies, the pathogenesis of this illness remains to be elucidated and effective therapies still await discovery. Recent findings suggest that treatment of amyloid accumulation may be insufficient for treating Alzheimer's disease, and raise the possibility that scientific and corporate research efforts have been too narrowly focused on the amyloid aspect of Alzheimer's disease. Alternative therapeutic strategies may ultimately prove to be key. In particular, targeting fundamental mechanisms of cell survival and maintenance that are disrupted in Alzheimer's disease may lead to cures of other neurodegenerative disease as well, as these downstream events are often reported in multiple age-dependent neurodegenerative disorders. Importantly, we have recently shown that deregulation of HDAC1 may be critically involved in CNS pathology that may be relevant to stroke/ischemia and Alzheimer's disease. Using an inducible p25/Cdk5 neurodegeneration mouse model, we have observed that the catalytic activity of the histone deacetylase (HDAC1), a key regulator of epigenetic modifications, was inhibited by p25/Cdk5. Loss-of-function of HDAC1 in neurons through multiple means resulted in the accumulation of DNA damage, cell cycle activity, and neuronal death. Conversely, overexpression of HDAC1 resulted in a rescue against p25-induced DNA damage and neuronal death. These results suggest a role for HDAC1 in the maintenance of DNA integrity and cell cycle suppression in adult neurons. Furthermore, overexpression of HDAC1 resulted in significant rescue against DNA damage and neurodegeneration in a rodent stroke model, demonstrating therapeutic potential for HDAC1 gain-of-function. As p25 accumulation, cell cycle reentry, and DNA damage appear to be features shared in multiple neurodegenerative conditions, it is anticipated that HDAC1 gain-of-function may be a valid therapeutic strategy against Alzheimer's disease, stroke, and other disorders. We propose here to develop a "proof-of-concept" of this strategy by identifying small molecules through a high-throughput screen that can increase the deacetylase activity of HDAC1. Preliminary results suggest the feasibility of these efforts and the ability of such small-molecule probes to prevent neurotoxicity. Given that the proposed strategy is significantly different from any of the previous therapeutic strategies that are undergoing development or have been abandoned, there is tremendous opportunity for these studies to have a high impact on human health. PUBLIC HEALTH RELEVANCE: The overall goal of this project is to discover and characterize selective chemical activators of the HDAC1 that can be tested for the ability to prevent neurotoxicity. Findings from this application may offer novel therapeutic strategy against Alzheimer's disease, stroke, and other neurological disorders.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is an age-related neurodegenerative disorder associated with severe memory impairments for which, currently, there is no cure. Although the role of beta-amyloid (A[unreadable]) in the disease is strongly supported by genetic evidence, the mechanism between A[unreadable] and neurodegeneration/memory impairments is far from clear. In combating AD, it is imperative that we expand our approach beyond the current focus upon amyloid pathology. Research into novel therapeutic approaches to combat the symptoms of AD has revealed beneficial effects of increased chromatin remodeling and gene expression. We have shown that small molecule inhibitors of histone deacetylases (HDACs) restore learning ability in the CK-p25 mouse model of AD even after severe neuronal loss has occurred. The class I histone deacetylase, HDAC2, has been shown to participate in the regulation of hippocampal-dependent learning and memory. HDAC2 binds to the regulatory elements of genes implicated in synapse formation and synaptic plasticity, and is upregulated in both the CK-p25 and the 5XFAD mouse models of AD. These findings have led to the idea that, during neurodegeneration, an altered epigenetic landscape, mediated by HDAC2 up-regulation, may repress the expression of gene products necessary for maintaining synaptic plasticity and memory functions. Thus, inhibition of HDAC2, even after the onset of neurodegeneration, can improve the function of surviving neurons. In the current application, we will test the hypothesis that a novel disease mechanism, involving HDAC2 mediated alteration of the epigenetic landscape, underlies the cognitive impairment and synaptic dysfunction of Alzheimer's disease. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) is an age-related neurodegenerative disorder associated with severe memory impairments for which, currently, there is no cure. We have shown that small molecule inhibitors of histone deacetylases (HDACs) restore learning ability in the CK-p25 mouse model of AD even after severe neuronal loss has occurred. The current application will test the hypothesis that a novel disease mechanism, involving epigenetics, underlies the cognitive impairment and synaptic dysfunction of Alzheimer's disease.

DESCRIPTION (provided by applicant): The looming specter of 13.8 million Americans with Alzheimer's disease (AD) by the year 2050 motivates us to expand our biomedical research paradigm outside of the typical cell line to rodent to human trials model. The disappointing performance of all AD drugs that have come to Phase III clinical trials to date also forces us to think more creatively about how to study the mechanisms that underlie neurodegeneration. The advent of human induced pluripotent stem cells (iPSCs) allows us to create disease models from patients with sporadic AD as well as from those with defined familial mutations. In addition, we can use cutting-edge genome editing techniques, such as the Crispr/Cas system, to introduce disease-associated mutations into the genome of otherwise healthy human derived pluripotent cells. In the past five years, neuroscientists have made incredible advances in the creation of different brain cell types, such as neurons, astrocytes, and oligodendrocytes, from human-derived pluripotent cells. What is lacking, however, is a human cellular model of neuroinflammation, a critical component of all neurodegenerative disorders, including AD. In the current application, we describe the creation of human microglia, the brain's immune cell, from patient-derived and control pluripotent cells. We propose a comprehensive set of experiments that will determine the role that known and novel AD-associated genes play in these cells using cutting-edge genome editing techniques combined with high-throughput functional assays, transcriptomic profiling, and high-resolution proteomics.

This project consists of engineering a system for producing selective expression of light-inducible molecules in targeted neuron population in non-genetically modified animals of any species. The result will be a set of reagents that will be made freely available to the scientific community through nonprofit repositories and service centers. This new set of tools will enable the study of neural circuitry with greater resolution, power, and throughput than is currently possible, allowing major advances to be made in understanding the organization of the complex neural systems underlying perception, cognition, and behavior. This increased understanding could also result in improved artificial intelligence and machine learning. Finally, the future direct application of the technology in human patients holds promise for potentially treating conditions such as Parkinson's disease and epilepsy, by allowing the selective activation or inactivation of distinct components of the compromised neural circuitry that is associated with these disorders.

Over the last decade, sophisticated genetic tools have been developed that allow control and monitoring of neuron electrical activity using light alone. "Optogenetics", as this area of technology has become known, is only useful if optogenetic molecules can be specifically expressed in functionally meaningful groups of neurons instead of broadly in all the diverse neuron types that are present in any brain region. This requirement has confined their use almost entirely to genetically modified (transgenic) mice and rats. The approach of using transgenic animals has three major disadvantages. First, the production and maintenance of transgenic rodents is very expensive. Second, even within transgenic rodents, it allows the optogenetic study and manipulation of only one or two cell types at a time, preventing powerful combinatorial experiments in which different neuron types are independently controlled within the same tissue. These combinatorial experiments will be critical for deciphering the complex interactions between cell types. Third, it restricts the experiments to rodents, preventing studies in other important taxa including primates, in which optogenetic experimentation during complex cognitive tasks would almost certainly provide major insights into the neural circuitry underlying cognition. This project aims to create engineered binding proteins that recognize selected endogenous proteins that will then act as scaffolds for assembly of transcription factors that will activate gene expression in specific neurons.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder associated with severe memory impairments for which, currently, there is no cure. Studies in human patients and mouse models reveal disease profiles that involve the downregulation of genes involved in neural functions, such as synaptic transmission, and the upregulation of immune response and inflammatory genes. Although there are a number of neural cell types implicated in AD risk and etiology, including neurons and different types of glia, the majority of studies thus far have utilized total tissue. While these have offered great insight into AD, the establishment of techniques for analyzing cell type-specific mechanisms underlying AD etiology is critically important. In the current proposal, we will utilize a combination of molecular techniques to label and isolate populations of specific cell types, including glia and neurons, and to generate cell type-specific epigenetic and transcriptomic maps from mouse models of neurodegeneration, postmortem human brain, and human adult stem cell-derived cultures. Using integrative bioinformatic analysis, we will determine the pathways disrupted during early and late neurodegeneration. We believe these studies will provide invaluable information about the gene expression and cellular programs that mechanistically underlie AD in the human brain, and will identify novel targets for therapeutic strategies.

DNA damage perturbs genomic stability and has been linked to age-associated cognitive decline, as well as to early stages of various neurodegenerative disorders including Alzheimer?s disease (AD), amyotrophic lateral sclerosis, and frontotemporal dementia (FTD). However, our mechanistic understanding of how DNA damage contributes to neuronal vulnerability and deterioration remains an unresolved, yet extremely important question. A major confounding factor is that the sources of damage that are most pertinent to neurodegeneration remain unknown and the precise mechanisms that connect genomic instability to neurodegeneration are poorly understood. In addition, it is unclear whether the deterioration of brain function results solely from a random accumulation of DNA damage throughout the genome, or whether there are ?hotspots? of damage that mediate this process. The goal of our research is to better understand the mechanisms underlying genomic instability in neurodegeneration and identify novel therapeutic targets to dampen this early pathological hallmark of neuronal vulnerability. We hypothesize that genomic instability is a major underlying mechanism of cognitive decline and neuronal vulnerability in AD and FTD. Towards testing this hypothesis, our specific aims are: 1) to identify genomic loci that are vulnerable to the accumulation of DNA damage, particularly DNA double strand breaks (DSBs) in mouse and human induced pluripotent cell (iPSC)-derived models of AD and FTD, 2) to determine the precise defects in DSB signaling/repair in mouse and human iPSC-derived models of AD and FTD, and 3) to identify modifiers that reduce DNA damage susceptibility in iPSC-derived neural cells from patients with familial AD and FTD using a novel high-throughput screening strategy. Our preliminary findings suggest that excessive DNA DSBs are an early pathological hallmark of neurodegeneration that can be modeled in both mouse and human systems. Obtaining increased mechanistic insight into the failure to respond to and/or repair DNA DSBs in the context of neurodegenerative mutations will broaden our understanding of how genomic instability contributes to decline in brain health and cognition, and provide novel avenues for early therapeutic intervention in neurodegeneration.

ABSTRACT The overarching goal of our LINCS project (U54HG008097) is to test the hypothesis that modulation of phosphorylation-mediated signaling events in response to perturbations can establish new cellular states by altering their phosphoproteomic and epigenetic landscapes. To achieve this goal, we propose performing mass spectrometry (MS)- based proteomic assays that specifically target quantitative readouts of phosphosignaling and chromatin modifications in cells on > 15,000 perturbational conditions. These perturbations will focus on modulation of signaling cascades and epigenetic marks by small molecules and genetic manipulations. We will study several different cellular model systems, including comprehensive studies of neuronal lineage differentiation starting from stem cells. We have established a center with the necessary infrastructure, pipelines, data management, and analytics required to perform the largest set of related experiments with MS proteomic read outs to date. We also employ next- generation MS acquisition technologies to establish a permanently minable MS data resource that will be accessible to the public. We contribute the resulting data and tools to the Library of Integrated Network-based Cellular Signatures (LINCS) program for the purpose of making connections among disparate perturbations through phosphoproteomic and chromatin modification signatures in concert with other data types to be contributed to LINCS by other centers. The resulting analyses help identify novel therapeutic opportunities and synergies, as dysregulation of phosphosignaling and epigenetic systems are two of the most common molecular etiologies identified in a growing number of genetic, developmental, and environmental diseases. In this supplement request we will study a unique model of Alzheimer's etiology derived from cell lines obtained from Down syndrome patients. We will test how cells with triplication of chromosome 21 respond differentially to kinase inhibitor stimuli from their otherwise isogenic disomic counterparts. We will measure aspects of signaling and epigenetics using proteomic analyses (P100 and GCP), and transcriptional responses using RNA-Seq and L1000 assays for maximum compatibility with the body of existing LINCS data.

Dementia is a major public health problem with substantial personal, social, and financial burden, affecting more than 47 million people worldwide, with no cure to date. The major types of dementia include Alzheimer?s disease (AD), Lewy Body dementia (LBD), and frontotemporal dementia (FTD), which show distinct and overlapping pathological, neurological, and cellular signatures, but their detailed molecular signatures remain uncharacterized. Here, we systematically profile the molecular signatures of AD, LBD, FTD, and healthy aging, at the single-cell level, across traits, individuals, brain regions, cell types, age, sex, and disease severity. We use genetic, epigenomic, and transcriptional profiles, generating a total of ~1.5 million genome-wide maps at the single-cell (sc) level using scRNA-seq and scATAC-seq across 768 post-mortem brain samples from the Religious Order Study and Memory and Aging Project (ROS MAP) cohorts. We analyze the resulting datasets in the context of genetic variation from whole-genome sequencing, and phenotypic variation from rich longitudinal profiling and cognitive evaluations, enabling us to discover genes, control regions, pathways, cell types, and brain regions playing causal roles in AD and ADRD, and how they vary across age, sex, and traits. The resulting datasets will help guide the search for new therapeutics, by providing detailed therapeutic targets, and the specific conditions where they are predicted to act.

Project Summary/Abstract Alzheimer's disease (AD) is a fatal neurodegenerative disease with a global prevalence close to 50 million people, which is expected to double by 2040. Finding an effective treatment for AD has proven difficult, as evidenced by numerous high profile Phase 3 clinical trial failures, most of which directly target the reduction of ß-amyloid. Thus, it is becoming increasingly urgent to develop new pharmacological strategies to combat AD. Drugs are twice as likely to successfully negotiate the drug development pipeline and obtain FDA approval when their targets are supported from human genetic studies of disease. Human genetic studies have revealed a critical role for microglia involvement in Alzheimer?s disease progression, and it has recently been discovered that the transcription factor PU.1 is a driver of the pro-neurodegenerative phenotype adopted by microglia during aging and disease. This proposal therefore aims to develop novel, newly-discovered PU.1 Inhibitory Modulators (PIMs) for preclinical development, with the long term goal of clinically testing the hypothesis that reducing PU.1 activity in microglia will safely delay the age of AD onset (AAO) in at-risk populations. The studies in this proposal leverage the interdisciplinary structure of the Neurodegeneration Consortium, a unique collaboration between basic science researchers and industry drug development veterans operating under a collaborative agreement to push forward novel therapeutics aimed at treating Alzheimer?s diseaes and other neurodegenerative diseases. Under Specific Aim 1, the in vivo safety and efficacy of PIMs will be determined in mouse models of AD. Under Specific Aim 2, parallel target engagement studies will be performed to identify the target of PIMs, and the identified targets will be used to develop assays to determine the efficacy of PIMs in AD and in ex vivo models. Under Specific Aim 3, selected PIMs will be optimized using PK/PD and ADMET screening to develop lead tool compounds into candidate compounds suitable for future Phase I studies. The combined biology, chemistry, and pharmacology expertise in the Neurodegeneration Consortium, spanning The University of Texas MD Anderson Cancer Center, the Massachussets Institute of Technology, and the Mt. Sinai School of Medicine, make this group of researchers ideally suited to execute the proposed aims.

Abstract Alzheimer?s disease (AD) is a devastating neurodegenerative disorder that leads to dramatic effects on the affected individuals and their families. While the characterization of the genetic contribution to AD and underlying molecular mechanisms have advanced the understanding of the disease in recent years, studies show that sex differences account for much of the observed differences in risk, progression, and severity across individuals. Here, we directly dissect the contribution of sex-specific variation down to the region- specific and cell-type-specific molecular basis by systematic profiling, computational integration, and experimental validation of the transcriptional, epigenomic, and genetic signatures across individuals, brain regions, and cell types. In Aim 1, we use genetic, epigenomic, and transcriptional profiles, generating millions of single-cell (sc) level maps using scRNA-seq and scATAC-seq across human and mouse samples of varying ages and genetic risk status. In Aim 2, we analyze the resulting datasets in the context of known AD genetic risk variation and underlying molecular mechanisms, enabling us to discover and converge variants, regulatory regions, genes, pathways, cell types, and brain regions to functional, causal mechanisms that drive sex-related differences. In Aim 3, we use our well-established mouse and iPSC models to test our predicted mechanisms with both high-throughput and cell-type specific assays. The resulting datasets, computational predictions, and experimentally-supported mechanisms will shed light on the sex-related differences of AD and will help deepen our understanding the disease in general as we develop more personalized therapeutic approaches in treating AD.