Wei-Dong Yao, Ph.D. - US grants

CRISP; Computer Retrieval of Information on Scientific Projects Database; DLG4; DLG4 gene; Dendritic Spines; Discs Large (Drosophila) Homolog 4; Discs Large Homolog 4; Dopamine Receptor; Funding; Goals; Grant; Head; Institution; Investigators; Mediating; Molecular; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nerve Cells; Nerve Unit; Neural Cell; Neurocyte; Neurons; PSD-95; PSD95; Postsynaptic Density-95; Property; Property, LOINC Axis 2; Receptor Signaling; Regulation; Research; Research Personnel; Research Resources; Researchers; Resources; SAP90; Scaffolding Protein; Source; Spinal Column; Spine; Synapses; Synaptic; Thinking; Thinking, function; United States National Institutes of Health; Vertebral column; backbone; dendrite spine; density; neuronal; postsynaptic; presynaptic density protein 95; trafficking

DESCRIPTION (provided by applicant): A central feature of addiction is compulsive drug taking, characterized by loss of control over drug use despite adverse physiological and social consequences. A prevailing theory in the neurobiology of cocaine addiction is that repeated drug exposure imposes persistent neurochemical and molecular changes, e.g. altered gene expression, in the mesocorticolimbic brain circuitry underlying reward. These cocaine-induced neuroadaptations may contribute to the complex manifestations of addiction but are poorly defined, especially in primates. The purpose of this application is to comprehensively investigate gene expression dysregulation within the reward circuit that may mediate compulsive cocaine taking, using nonhuman primate models of moderate or excessive cocaine self-administration. Monkeys self-administering cocaine will be yoked to monkeys passively receiving cocaine or saline injections for different exposure periods that reflect initial stages of addiction, or "recreational", drug use and more chronic cocaine use associated with addiction. A similar study will be conducted in which monkeys self-administer a non-drug reinforcer (sucrose pellets). We postulate that a dysregulation of the dopamine (DA) and glutamate (Glu) systems is associated with chronic cocaine taking. We further postulate that ensemble adaptations in multiple brain structures within the reward circuit as a consequence of DA/Glu dysregulation may accompany chronic cocaine taking. We specifically will investigate adaptations in mesocorticolimbic DA and Glu systems induced by initial and chronic cocaine self-administration by characterizing changes in key components of DA and Glu systems using in situ hybridization, quantitative PCR, and immunoblotting (Aim I). Aim II will define gene expression patterns at the genome level associated with initial and chronic cocaine self-administration, using a newly developed Rhesus Macaque Gene Chip. Successful completion of the proposed research will provide an integrated assessment of the DA and Glu systems, identify individual novel dysregulated gene candidates, and provide a near genome view of transcriptome changes associated with the reinforcing effects of cocaine in multiple brain structures within the reward circuit. The outcome of the research should deepen our understanding of the molecular and cellular basis of compulsive drug taking.

CRISP; Computer Retrieval of Information on Scientific Projects Database; Condition; Corpus Striatum; Corpus striatum structure; DLG4; DLG4 gene; Discs Large (Drosophila) Homolog 4; Discs Large Homolog 4; Funding; Grant; Institution; Investigators; Mediating; N-Methyl-D-Aspartate Receptors; NIH; NMDA Receptor-Ionophore Complex; NMDA Receptors; National Institutes of Health; National Institutes of Health (U.S.); Nerve Cells; Nerve Degeneration; Nerve Unit; Neural Cell; Neurocyte; Neurologic; Neurological; Neuron Degeneration; Neurons; PSD-95; PSD95; Play; Postsynaptic Density-95; Receptors, N-Methylaspartate; Research; Research Personnel; Research Resources; Researchers; Resources; Role; SAP90; Source; Striate Body; Striatum; Testing; United States National Institutes of Health; excitotoxicity; neural degeneration; neurodegeneration; neuron toxicity; neuronal; neuronal degeneration; neuronal toxicity; neurotoxicity; presynaptic density protein 95; social role; striatal

3,4-Dihydroxyphenethylamine; 4-(2-Aminoethyl)-1,2-benzenediol; Ammon Horn; Brain region; CRISP; Computer Retrieval of Information on Scientific Projects Database; Cornu Ammonis; Dopamine; Funding; Grant; Hippocampus; Hippocampus (Brain); Hydroxytyramine; Institution; Investigators; Long-Term Potentiation; Memory; Molecular; NIH; National Institutes of Health; National Institutes of Health (U.S.); Prefrontal Cortex; Research; Research Personnel; Research Resources; Researchers; Resources; Role; Site; Source; Synaptic plasticity; United States National Institutes of Health; hippocampal; social role

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. RNA interference (RNAi) is an evolutionarily conserved process of sequence-specific post-transcriptional gene silencing. Viral-mediated RNAi allows rapid knockdown of endogenous proteins in post-mitotic cells, including neurons and has great potential in facilitating understanding the neurobiological basis of and therapeutic development against many psychiatric and neurological disorders. The goal of this project is to develop a RNAi-based gene silencing system in rhesus macaque.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Functional activity studies have established prefrontal cortex (PFC) as a critical site for the formation and storage of permanent memories, which likely requires synaptic plasticity in the PFC. Unlike other brain regions, e.g. the hippocampus, the cellular and molecular mechanisms underlying PFC plasticity are poorly understood. This project investigates the role of dopamine in prefrontal synaptic plasticity.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Dysfunction of the neuronal microcircuits in the prefrontal cortex is believed to underline the pathogenesis of some major neuropsychiatric disorders, such as schizophrenia. Generation and maintenance of persistent neuronal activity is one of essential features of these prefrontal circuits underlying many prefrontal functions, including working memory. The aims of this project are to investigate 1) the basic synaptic transmission properties in prefrontal microcircuits, 2) synaptic plasticity, especially spike timing-dependent plasticity (STDP), and its interaction with persistent neuronal activity, 3) the modulatory effects of dopaminergic input and underlying molecular mechanisms.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. PSD-95 is a prototypical scaffold enriched in the postsynaptic density (PSD) of excitatory synapses and plays an important role in localization of glutamate receptors in the synapse and synaptic plasticity. Mechanisms regulating PSD-95 levels in the synapse are not well understood. The aim of this project is to identify novel PSD proteins involved in regulation of PSD-95 and elucidate their roles in synaptic development, function, and plasticity.

DESCRIPTION (provided by applicant): The goal of this NCRR SIG grant proposal is to acquire a state-of-the-art laser-scanning confocal microscope to support research of 14 NIH-funded Investigators (PIs) at the New England Primate Research Center (NEPRC), a major research facility at Harvard Medical School (HMS). NEPRC is a NCRR supported national primate center that pursues a mission that integrates research, training, and services in biology and medicine, focusing on human health and primate biology. The existing multi-user confocal system, a Leica TCS SP1 purchased in 1998, has served as a workhorse for us over 10 years and provided outstanding service to the broad research community at NEPRC. While it was state-of-the-art in the 1990's, this heavily- used, aging system no longer meets our needs in temporal and spatial resolution, photosensitivity, imaging quality, and versatility. This system is incapable of supporting many novel and powerful applications available in today's molecular/cellular/system imaging applications and is not upgradeable. We present research projects from 14 investigators (11 Major Users and 3 Minor Users) at NEPRC and affiliated institutions. These projects represent a broad array of research interests ranging from molecular and cellular neurobiology to neurological and psychiatric disorders, from microbiology to AIDS pathogenesis, and from molecular virology to cancer. Each major user has in common a strong history of NIH funding (they receive in excess of $10M in direct NIH funds per year from 10 categorical institutes) and a need for advanced imaging technology. We propose to purchase a Leica TCS SP5 405UV Spectral Confocal Microscope system with an inverted microscope, 8 laser lines with AOBS, a UV 405 laser, prism spectrophotometer detectors and easy-to-use advanced software with FRAP and FRET controls, all of which are essential to the projects proposed by the major users. Compared to other premiere systems on the market, the Leica TCS SP5 offers the best overall package in quality, options, service, and price. It also permits enormous power and flexibility and accommodates many novel applications that fit into the needs of diverse research interests at NEPRC. The system will be housed in the NEPRC imaging core facility, maintained by a highly experienced core staff, and supported by a NCRR base grant. The new confocal system would be essential to the NIH-sponsored research of the Major and Minor users at NEPRC, and greatly facilitate the overall mission of the NEPRC as a national center for research, service, and training. The research to be performed with this system will have direct impact on understanding the molecular and cellular basis of a range of neurological, psychiatric, and infectious diseases.

DESCRIPTION (provided by applicant): Drug addiction is a chronic brain disease characterized by uncontrolled drug taking, craving, and relapse. Addictive drugs invariably induce non-physiological DA signal that likely interferes with ongoing motivational and associative learning behaviors, modify reward circuits via plasticity mechanisms similar to that underlie these behaviors, and alter reactivity of these circuits with DA, perpetuating use of a drug. A particularly important region in the dopaminergic reward circuitry is the prefrontal cortex (PFC), which mediates executive control of motivation and choice and is implicated in directing addictive behaviors. Alterations in glutamatergic plasticity are hypothesized to promote the compulsive character of drug seeking in addicts and hinder extinction of drug use memories, promoting relapse. Unlike primary sensory cortices, PFC circuits are to some degree refractory to experience scalping but are readily modified by drugs, suggesting unique plasticity mechanisms that show increased dependence on DA in this associative cortex. Precise mechanisms by which DA drives synaptic plasticity in PFC are poorly understood. In particular, it is unclear (i) how glutamatergic synaptic modifications can occur in native circuits tightly controlled by GABAergic inhibitory tone, (ii) what precise roles DA might play in enabling synaptic plasticity as suggested by behavioral studies, and (iii) how addictive drugs modify PFC circuits and their reactivity to DA, resulting in an addicted circuitry. Our recent studies indicate that a brief phasic DA is necessary to enable spike-timing dependent long-term potentiation (t-LTP) in native PFC circuits under conditions of intact GABAergic inhibition. This enabling requires a cooperation between D1-class receptors (D1Rs) in excitatory circuits and D2-class receptors (D2Rs) in inhibitory circuits, whereby D2R activation gates t-LTP induction by suppressing local GABAergic inhibition and D1R activation controls the timing window for t-LTP induction, respectively. Our results reveal a previously unrecognized circuit-level mechanism by which DA receptors in separate microcircuits cooperate to drive Hebbian synaptic plasticity. The goals of this R01 application are to define the molecular, synaptic, and signaling details in interconnected PFC excitatory (Aim 1) and inhibitory (Aim 2) circuits that permit DA to empower synaptic modifications in the PFC. We will also investigate how repeated cocaine exposures in vivo alter the t-LTP induction and dopaminergic teaching rules in PFC synapses (Aim 3). A combination of slice electrophysiological, molecular, biochemical, and morphological approaches will be employed. These studies address fundamental issues concerning modifications of PFC inhibitory and excitatory microcircuits, and the roles of DA reward signal in these processes. Our studies will also provide key insights into how addictive drugs may erode intrinsic rules governing associative plasticity and usurp the prefrontal reward circuitry. The information obtained will advance our knowledge of the reward circuitry plasticity mechanisms and facilitate understanding and treatments of addiction.

? DESCRIPTION (provided by applicant): Altered assembly, function, and plasticity of synapses and neural circuits underlie cognitive, memory, and emotional deficits of essentially all neuropsychiatric and neurological diseases. A well-investigated mechanism that regulates synaptic protein turnover and synapse remodeling is the conventional ubiquitin-proteasome pathway, by which polyubiquitin chains conjugate to protein substrates (likely through lysine 48 (K48) of ubiquitin) and tag them for proteasomal degradation. A vastly overlooked ubiquitin modification in the central nervous system (CNS) is K63-linked polyubiquitination, an unconventional linkage mechanism generally believed not to target proteasomal degradation; rather, it regulates protein scaffolding, trafficking, and activity. Despite its recognized importace in cellular signaling mechanisms that mediate innate and adaptive immunity, virtually nothing is known about the role of this proteasome-independent polyubiquitination process in the CNS, especially synapses. Nonetheless, K63-linked ubiquitination is the second most abundant linkage in the rat brain, only slightly behind the K48 linkage. In addition, emerging evidence points to a link between K63-linked ubiquitination and a number of brain disorders. Thus, there is a need to investigate the mechanisms and roles of this nonproteolytic polyubiquitin topology in neurons and at synapse. Our preliminary studies indicate that K63-linked polyubiquitination is a fundamental mechanism regulating synapse assembly and plasticity and identify the postsynaptic scaffolding protein PSD-95 as the first substrate. We also identify a PSD-associated, K63-linkage-specific enzyme machinery that controls PSD-95 ubiquitination at synapses. The goals of this R01 application are to define the molecular details and functional consequences of PSD-95 K63 polyubiquitination (Aim 1), to delineate the role of K63 polyubiquitination in synapse development, function, remodeling, and plasticity (Aim 2), and to characterize the behavioral alterations in mice lacking K63-linkage-specific E3 ubiquitin ligase TRAF6 and deubiquitinase CYLD (Aim 3). A combination of molecular, biochemical, electrophysiological, and behavioral approaches will be employed. The proposed studies represent a fundamentally important conceptual breakthrough that opens up new avenues for investigation of synapse and circuit function and plasticity. The project has the potential to uncover new paradigm-shifting principles that govern neuro-immune interactions, which may contribute to abnormal brain wiring in neurodevelopmental disorders (such as schizophrenia and autism spectrum disorders) and neural circuit repair in the adult brain following injury. The information obtained will facilitate development of novel treatment strategies for these brain 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.

Frontotemporal dementia (FTD), the second most common form of dementia after Alzheimer?s disease (AD), is caused by atrophy of frontal and/or anterior temporal lobes. FTD is characterized by changes in personality, loss of empathy, apathy, disinhibition, and language disability at early-mid stages, and general cognitive deteriorations at later stages. FTD is linked clinically, pathologically, and genetically to amyotrophic lateral sclerosis (ALS) and understanding FTD pathogenic mechanisms also has significant implications for AD. Molecular and cellular mechanisms underlying FTD are poorly understood. Up to 50% of FTD are familial and associated with mutations of at least 15 genes of diverse functions, suggesting a strong genetic component. Remarkably, at least 10 of these genes are involved in autophagy, a conserved cell quality- control process that delivers cytoplasmic contents to lysosomes for degradation. Autophagy has emerged as a central mechanism in FTD/ALS and other major neurodegenerative diseases. However, it remains enigmatic how autophagy is dysregulated in FTD/ALS and how exactly autophagy dysfunctions cause the diseases, presenting a major hurdle and knowledge gap in development of autophagy-based therapeutic strategies. Recently, three rare variants of the CYLD gene, predicted to have high pathogenic potentials, are identified in FTD/ALS patients, placing CYLD as the newest member of the FTD/ALS-causing gene family. CYLD encodes a Lys63-specific deubiquitinating enzyme and interacts with several FTD gene products that regulate autophagy flux, suggesting a potential role for CYLD in autophagy related to FTD. CYLD is known as a tumor suppressor linked to familial cylindromatosis (skin tumors in head and neck areas) and immune signaling, but its roles in neurons and synapses are largely unknown. Our published and unpublished studies indicate that CYLD is a synapse-enriched Lys63-specific deubiquitinase that has a major role in synapse maintenance, function, and plasticity through regulation of neuronal autophagy. The goals of this R21 application are to delineate the molecular characteristics of FTD-linked CYLD variants and explore their potentials to induce FTD-related pathologies, synapse loss and dysfunctions, and behavioral impairments. Our study represents the first attempt to investigate the role of a new disease gene in FTD pathogenesis. The proposed studies are fundamentally important and highly significant because they have the potential to uncover novel genetic and molecular mechanisms and treatment strategies for FTD/ALS and related dementia.

Frontotemporal dementia (FTD) is the leading dementia before the age of 65 and the second most common form of dementia after Alzheimer?s disease (AD). There is no cure. FTD is caused by focal but progressive atrophy of frontal and/or anterior temporal cortices that leads to changes in personality, apathy, loss of empathy, disinhibition, and language disability at early-mid stages, and general memory and cognitive deteriorations requiring a full-time care at later stages. Thus, synaptic and circuit dysfunctions underlying behavior impairments may precede massive neuronal cell loss and disability. However, molecular mechanisms underlying disease initiation and early symptoms as well as disease progression are not well understood. FTD is linked clinically, pathologically, genetically, and mechanistically to amyotrophic lateral sclerosis (ALS). Up to 50% of FTD are familial and associated with mutations of at least 15 genes of diverse functions. Remarkably, at least 10 of these genes are involved in autophagy, which has emerged as a central mechanism in FTD/ALS. However, it remains enigmatic how autophagy is dysregulated in FTD/ALS and how exactly autophagy dysfunctions cause the diseases, presenting a major hurdle and knowledge gap in development of autophagy-based therapeutic strategies. Recently, a gain of function mutation in the CYLD gene is identified in FTD/ALS patients, placing CYLD as the newest member of the FTD/ALS-causing gene family. CYLD encodes a Lys63-specific deubiquiting enzyme and interacts with several FTD gene products, including p62/SQSTM1, Optineurin, and TBK1, suggesting a potential role for CYLD in autophagy related to FTD. CYLD is best known as a tumor suppressor linked to familial cylindromatosis and immune signaling, but its roles in neurons and synapses are largely unknown. Our published and unpublished studies indicate that CYLD is an abundant Lys63-specific synaptic deubiquitinase that has a major role in synapse maintenance, function, and plasticity through regulation of neuronal autophagy. The goals of this R01 application are to define the molecular details and functional consequences of CYLD-dependent autophagy (Aim 1), to generate an inducible transgenic mouse model and delineate the role of FTD-causing mutation CYLDM719V in FTD pathogenesis (Aim 2), and to validate the pathogenic role of CYLDM719V in FTD and explore therapeutic strategies in human induced pluripotent stem cell (iPSC)-derived cortical neurons (Aim 3). Our study represents the first attempt to investigate the role of a new disease gene in FTD pathogenesis. Our proposed studies are fundamentally important and highly significant because they have the potential to uncover novel pathogenic mechanisms and treatment strategies for FTD and related neurodegenerative diseases.