Zhiping Pang - US grants

DESCRIPTION (provided by applicant): This is an exploratory/developmental (R21) application under the Cutting-Edge Basic Research Awards (CEBRA) program. The pathogenesis of drugs associated with abuse behavior, including nicotine and alcohol addictions, remains elusive in humans because studies of the human brain are limited to functional brain imaging and post-mortem analysis. These types of analyses make it difficult or impossible to prove hypotheses directly since the system usually cannot be manipulated or sufficiently controlled. A large number of genetic variants have been identified to be risk factors for addictive behavior in human, however, little is known about how these genetic variations impact the development of addictive behavior in humans. Recent advances in stem cell biology allow construction of induced pluripotent stem cells (iPSC) from adult cells derived from addicted individuals carrying identified genetic variants and provide possibilities for developing cell-based models of addiction. Addictive behavior in human is not only related to cellular level modifications in a specific cell type in the brain but it also affects neuronal function such as synaptic plasticity at the neurocircuitry level. However, there are currently no such in vitro neurocircuitry models that have been established using human neurons. We hypothesize that neurons derived from subjects with risk-associated genetic variants will desensitize reward circuit modulation in an in vitro mini-neurocircuitry model. By using a compartmentalized culturing system, we propose to construct a mini-neurocircuitry model mimicking mesolimbic nucleus accumbens (NAc) neurons and their synaptic inputs. Cellular and synaptic phenotypes of neurons derived from addictive patients will be investigated under the context of neurocircuitry and compared with wild-type controls. This mini-neurocircuitry model will be essential to identify mechanisms underlying risk-associated gene variants and addictive behavior. It will also serve to develop and screen novel interventions for drug abuse therapies.

The brain encodes information in the form of electrical impulses called action potentials that are continuously exchanged between neurons. As brains age, the transmission of Action Potentials becomes less efficient. It is not understood why this occurs, but one possibility is that over time, neurons accumulate highly toxic molecules known as reactive oxygen species (ROS). There is support for the idea that ROS oxidize potassium channel proteins whose function is to permit movement of potassium ions through the membranes of neurons. Potassium ion movements are involved in the propagation of action potentials by neurons. This project will directly test the hypothesis that oxidation of potassium channels by ROS leads to cognitive impairment by hindering the transmission of Action Potentials. The PI will integrate research activities and outcomes into formal lectures on the neuroscience of aging. The project will provide research training opportunities for undergraduate, graduate, underrepresented and summer students. The Principal Investigator will present a series of lectures to middle and high school students as part of the Science, Medicine and Related Topics (SMART) program, whose mission is to advance the understanding and appreciation of science among underrepresented students.

The KCNB1 potassium channel is abundantly expressed in the brain and is susceptible to oxidation by ROS. When a cysteine residue is replaced with an alanine (C73A), oxidation is abolished. Based on this observation, a transgenic mouse expressing the non-oxidable KCNB1 variant (C73A) has been constructed. The effects of ROS on KCNB1 channels and the impact of this process on cognitive function will be determined by studying cognitive function in aging C73A mice. Behavioral tests will be performed to see whether the lack of oxidation of KCNB1 improves the ability of mice to learn and remember new tasks. Changes in behavior will be correlated with changes in the electrical activity of the brain which will be recorded using state of the art electrophysiological techniques. Further, it will be determined whether the naturally occurring antioxidant, hydrogen sulfide, which is synthesized by neurons, can prevent oxidation of KCNB1 channels. It is expected that aging mice that harbor the C73A variant will exhibit less cognitive impairment and more normal electrical activity in cortex and hippocampus, two regions of the brain that are most affected by the activity of KCNB1.

? DESCRIPTION (provided by applicant): Alcoholism is a serious health and socioeconomic problem in the U.S. Understanding how alcohol produces reward, motivates further consumption, and eventually leads to addiction is necessary to design effective treatments for alcohol use disorders (AUDs). Synaptic transmission mediates all of these behaviors, however, little is known about the effect of alcohol on synaptic transmission in the context of human neurons. The single nucleotide polymorphism (SNP) rs1799971 (OPRM1 A118G) produces a non-synonymous amino acid substitution in the mu-opioid receptor (MOR), in which Asparagine 40 (MOR N40) is replaced with Aspartate (MOR D40), and is associated with AUDs in specific ethnic groups. Importantly, Naltrexone, a nonselective MOR antagonist, has potent therapeutic effects in alcoholic individuals with MOR D40. We generated human neurons from 7 subjects carrying either homozygous MOR N40 or MOR D40. Our preliminary data suggest that human neurons carrying D40 show defective re-sensitization after MOR activation by DAMGO ([D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin), suggesting defective trafficking of MORs. Supporting this, bioinformatic analyses and mouse models of human MOR N40D suggest that D40 disrupts an N-glycosylation site on MOR. However, the mechanism by which MOR protein trafficking defects affect the interaction between ethanol and opioids is not known. It is particularly important to reveal the molecular mechanisms underlying the function of N40D MOR variants in their native neuronal context because previous studies performed in heterologous systems have revealed inconclusive and confusing results. Moreover, a species-specific trafficking mechanism of MORs has been suggested. The objective of this proposal is to study the impact of alcohol and opioid signaling in both mouse and human neurons carrying both the N40 and D40 MOR allelic variants, focusing on the synaptic mechanisms that likely underlie behavior. The central hypothesis is that defective D40 MOR trafficking results in an altered effect of the interaction between alcohol and opioids on synaptic function in the reward neurocircuitry. We will first examine the effect of alcohol on synaptic function in a defined neurocircuitry composed of human neurons carrying these gene variants. Next, we will use a mouse model of human N40D, and study the synaptic mechanism in the reward neurocircuitry, i.e. the midbrain ventral tegmental area (VTA), in relation to MOR function and ethanol. The proposed research is innovative, because we will combine recent developments in stem cell biology, the state-of-the-art synaptic physiology, and novel microfabrication technologies to directly probe the impact of alcohol and opioid signaling on synaptic function in both mouse and human neuronal networks carrying OPRM1 gene variants. We expect to unravel a species- and cell type-specific mechanism of MOR N40D variants that may provide novel information for understanding AUDs.

PROJECT SUMMARY Trisomy 21 (T21, a.k.a. Down syndrome) is the most common genetic form of intellectual disability, and is caused by inheriting three copies of chromosome 21 (HSA21). Animal models of T21 have demonstrated a number of synaptic aberrations. Accumulating evidence indicates that the 3' untranslated region (3'UTR) of mRNA plays important roles in mRNA metabolism in neurons, including mRNA stability, translation, and localization. The 3'UTR is a hotbed for cis elements targeted by microRNAs (miRNAs) or bound by RNA- binding proteins (RBPs). Both miRNAs and RBPs have been implicated in spinogenesis, dendritic arborization, and synaptogenesis. Interestingly, owing to alternative cleavage and polyadenylation (APA), neuronal 3'UTRs are much longer than those in other cell types, adding another layer of post-transcriptional gene regulation in neuronal cells. However, little is known about the role of 3'UTR in the etiology of T21, and how APA is regulated during neurogenesis of T21 cells has never been explored. The objectives of this project are to 1) examine how 3'UTR isoforms are expressed during neurogenesis of normal and T21 cells; and 2) how post- transcriptional regulation is executed via 3'UTRs in normal and T21 neurons.

Abstract Synaptic transmission is mainly mediated by classical neurotransmitters such as glutamate and g-amino butyric acid (GABA) which transduce fast information flow in the brain. This process is tightly regulated by neuromodu- lators including monoamines and neuropeptides. Among neuromodulators, neuropeptides in particular, have been difficult to study because they are often chemically inert (non-oxidizable) and typically activate 7 transmem- brane G-protein coupled receptors (GPCRs) which rely on delayed (seconds to minutes) second messenger signaling, precluding their study by conventional electrophysiology or with oxidizable probes. Moreover, endog- enous neuropeptides are typically sorted from large polyprotein precursors into specific pools of dense core vesicles (DCVs), suggesting the need for a detection strategy that does not interfere with the biogenesis and native sorting of endogenous DCVs. As a result, there is currently a lack of suitable tools for studying the spatial and temporal dynamics of neuropeptide release and peptidergic neurocircuitry. Toward addressing this need, we propose to develop a new family of genetically-encoded optical sensors, termed Chimeric Detectors for Neu- ropeptide Release (CDNRs), by harnessing the unique structural signatures of Class B (Secretin-like) GPCRs that exclusively recognize peptides as their native ligands. Based on recently solved protein structures and the availability of new robust genetic models to apply and validate our approach, we will specifically focus on gluca- gon-like peptide-1 (GLP-1) and corticotropin-releasing hormone/factor (CRH/CRF) which have important func- tions in complex neurobehaviors such as feeding and stress. To validate CDNRs for detection of neuropeptide release, we will express CDNRs in defined GLP-1 and CRF circuitry using state-of-the-art viral mediated gene transduction in specific genetically modified mouse models and perform high-resolution optical recording to de- tect release of endogenous GLP-1 and CRF, both ex vivo in brain slices and in vivo in behaving animals. Suc- cessful implementation of this work will deliver a set of novel and well validated genetically-encoded CDNRs that can be immediately applied to dissect the peptidergic circuitry of GLP-1 and CRF in the brain. Moreover, this will also lay a conceptual and technical foundation for the future development of detectors for several other neuro- peptides (PACAP, VIP, CGRP and secretin) included among peptide-binding class B GPCRs. !

Abstract/Project Summary In this project, we request funds to acquire an upright Bruker NeuraLight 3D spatial laser modulator (SLM) Ultima multiphoton microscope to enable simultaneous multiphoton imaging and 3D holographic optical stimulation. The instrument will allow simultaneous deep brain, decoupled, two-photon functional imaging of neuronal activity and SLM holographic optogenetic stimulations to study and understand neural connectivity and how neural networks control behavior. The instrument will be housed at the Child Health Institute of New Jersey (CHINJ) at Rutgers Robert Wood Johnson Medical School (RWJMS) to support research in unraveling the molecular, synaptic, and cellular mechanisms underlying mental disorders in a cell type- and circuity-specific manner. This instrument will be the first of its kind on the Rutgers Biomedical and Health Sciences (RBHS) and Rutgers New Brunswick/Piscataway campuses and will fill a critical void in available technologies to multiple investigators funded by NIMH at Rutgers. The core users of the system will be prioritized to NIMH-funded research laboratories with expertise in developing novel optical sensors, and studying fear, depression, and autism and Tourette syndromes, but will also be available for use by other NIH-funded researchers. This cutting-edge instrumentation will meet a critical need in the dramatically increased demand for deep tissue imaging with cell type and circuitry- specific optogenetic manipulations, essential for understanding complex brain disorders and behaviors, which cannot be met by any other systems currently available on campus. Recent developments in optics technologies, specific opsins and genetically encoded neuronal activity reporters including GCaMPs (calcium), Voltron & ASAP (voltage), dLight or GRAB-DA (dopamine), as well as Reporters for Transmission mediated by GPCRs (RTGRs) which we are currently developing, have made these research paradigms possible. Importantly, we have obtained strong commitments from the CHINJ, Rutgers-RWJMS, and Rutgers-School of Arts and Sciences for this application to acquire this state-of-the-art upright multiphoton microscopy system, including designated space, funding for additional instrumentation costs, and long-term support for an extended service contract and maintenance for the system. The principal investigator and a senior lab research associate, along with local technical experts, will be responsible for all user training and routine daily maintenance, as well as providing user assistance, consultation on experimental design and advice on system configuration and optimal system use. An advisory committee composed of members with strong expertise in biophotonics has been formed to ensure proper and efficient use of the facility. We anticipate that the addition of this system will not only greatly strengthen current NIMH-funded projects but will also allow users to explore new questions previously not addressable due to the absence of a platform able to image deeply into samples with high speed neuronal activity manipulations. Therefore, this instrument will play a critical role in promoting new discoveries in neuroscience and help towards devising novel therapeutics for treating mental disorders.

Modified Project Summary/Abstract Section Rare loss-of-function (LoF) mutations in SETD1A are strongly associated with schizophrenia (SZ), a debilitating mental disorder affecting 1% of the population, and other severe neurodevelopmental disorders. SETD1A encodes a component of the histone methyltransferase complex producing mono-, di, and trimethylated histone H3 at Lysine 4 (H3K4). H3K4 trimethylation (H3K4me3) and H3K4me1 are epigenomic marks of active gene transcriptional promoters and enhancers, respectively. Interestingly, histone methylation has also been suggested as one of the most enriched gene pathways in common variant-based genome-wide associations studies (GWAS) of major psychiatric disorders. Furthermore, a recent mouse model with heterozygous knockout of SETD1A exhibited working memory deficits and showed transcriptional changes that overlap with those implicated in neurodevelopmental disorders, however, seemingly independent from a H3K4me3 mechanism. Therefore, it remains largely unclear whether and how SETD1A causes SZ-relevant molecular and cellular changes in a human brain. Our central hypothesis is that human induced pluripotent stem cell (hiPSC)-derived neuronal cells and cortical organoids recapitulate key SZ-relevant epigenetic, molecular and cellular properties of SETD1A LoF in the human brain. Using CRISPR/Cas9 gene editing, we have generated isogenic hiPSC lines carrying heterozygous LoF mutations (in exon 4 and exon 16, on different genetic backgrounds) of SETD1A. Preliminary results showed that mutant lines were defective in cortical organoid development with premature neuronal differentiation at early developmental stages. Furthermore, morphological, electrophysiological and transcriptomic analyses of hiPSC neurons carrying SETD1A LoF mutation showed defective synaptic neurotransmission. Interestingly, genes showing differential expression in both 3D cortical organoids and 2D cultures from mutant lines are enriched for common GWAS risk variants of SZ and other neuropsychiatric disorders/traits, suggesting possible convergent pathways shared by SETD1A LoF and common GWAS risk variants of major psychiatric disorders. Leveraging our respective expertise in hiPSC models and neurogenesis, synaptic physiology and functional genomics within our team, we propose to characterize the molecular and cellular mechanisms underlying the deficits associated with SZ-associated LoF mutations in SETD1A in human neural systems. We will identify the cell-type-specific and developmental stage-specific cellular and molecular phenotypes associated with SETD1A LoF in cortical organoids, and then investigate the synaptic phenotype(s) of SETD1A LoF mutations in human neurons and associated transcriptome changes. The proposed study will enable us to perform a well-controlled assessment of the impact of SETD1A LoF mutations on the molecular and cellular mechanisms underlying deficits in early neurodevelopment and synaptic properties.

Abstract Deficits in neurodevelopment and neuro-neuronal communications lead to mental disorders including autism spectrum disorder and schizophrenia in humans. The progress in understanding the pathophysiology of mental disorders is hampered by the lack of an integrative understanding of molecular, morphological and functional properties of diverse cell types in human brain. While 3D cortical organoids derived from human induced pluripotent stem cell (hiPSC) have been used to model neuropathology associated with virus infections and neuropsychiatric disorders, it is still unclear whether the early brain developmental process can be faithfully recapitulated by hiPSCs-based cortical organoids. Single-cell RNA sequencing of tens of thousands of cells of human cortical organoids has provided an unprecedented opportunity to dissect the spatial and temporal mechanism in early neuronal development in a cell type-specific manner. Such an approach has enabled the classification of many neural types in several species and organoids based on transcriptomic profiles, which are remarkably similar to the cellular compositions in human early brain development. Despite the advances in single cell transcriptomics, the electrophysiological properties as well as morphological features of different types of human neurons in brain organoids remain elusive. The labor-intensive nature of classical patch clamp electrophysiology and the technical difficulties in recording from a heterogeneous population of neurons at different stages of maturation had limited the abundance of functional data in human neurons. Because electrophysiological phenotypes, contributed by morphological features, are governed by distinct membrane ion channels and receptors, we hypothesize that electrophysiological (and possibly morphological) features of human neurons can be predicted by single cell transcriptomic profiles. The primary goal of this exploratory project is to establish a cell-census map based on electrophysiological, morphological and single cell transcriptomic profiles in a hiPSC-3D cortical organoid model and to develop a transcriptomic algorithm for predicting cell morphology-electrophysiology of human neurons. To achieve this goal, we propose: 1) to build a cell census map of neural subtypes of human 3D cortical organoids with functional annotation at single cell resolution; 2) to use using single cell transcriptomic profiles to predict the morphological and functional properties of cell types in human 3D cortical organoids. This exploratory project will allow us to develop a database to integrate single cell transcriptomes with cellular properties including electrophysiology and morphology profiles which enable predictions of neuronal functions in brain development, health and disease based on transcriptomic data. This study has enormous potential to enable future studies to ascertain the functional properties of neurons in organoids based on transcriptomic data, thus facilitating the modeling of early brain development in health and disease.