Jerold Chun - US grants

Neuropsychiatric development disorders involving the neocortex have a molecular basis that is virtually unknown. The long-term goal of this work is to identify key molecules used in normal development that will serve as a foundation for understanding these disorders. This proposal uses a novel strategy based upon my ability to generate stable cell lines with neuronal phenotypes from neuroblasts that are destined for specific layers during cortical neurogenesis. Although the characterization of these lines is incomplete, the preliminary data indicate the feasibility of generating cell lines based on cortical layer and demonstrate the advantages that these lines offer for studying the genes that underlie neocortical development. A most basic developmental question about the formation of layers in the neocortex is whether the adult neurons composing a layer are genetically committed prior to positioning within that layer, or are initially plastic, molded by subsequent environmental interactions within the neocortex to form the layer. Towards answering this question, I will test the hypothesis that new cell lines, derived from neuroblasts destined for a cortical layer, express novel genes that specifically identify young neurons destined for the same layer in vivo. Neuronal lines will be generated with a novel procedure based on targeting neuronal progenitor cells with retroviruses, resulting in the cellular expression of oncogene combinations. Using this procedure I've produced over 35 stable cell lines from the murine neocortex. Preliminary data on two of these lines have shown that they are phenotypically stable by neurofilament and neuron specific enolase expression and are clonal. A key advantage of a cell line is that mRNAs, that are easily isolated in large quantities, are a vastly less complex population compared with intact brain. This will allow the detection of even single-copy mRNAs and I will use this advantage to generate unbiased nucleotide probes against mRNAs selected by subtractive techniques; these probes have no inherent bias for molecular motifs that might identify a neuron in vivo. The complementary approach will bias probes for a class of molecules shown to be important for mammalian muscle and Drosophila nervous system differentiation, the helix-loop-helix transcription factors. Unbiased and biased probes will be screened on developing neocortical tissue by northern blots and in situ hybridization. Probes labelling discreet neocortical populations of neurons will be characterized by cDNA and genomic cloning of the genes followed by sequence analyses.

G protein; laboratory mouse

DESCRIPTION Neurological disorders that stem from cortical neuron dysfunction result in part from the disruption of normal developmental processes within the cerebral cortex. The long-term goal of this proposal is to understand how cortical neurons are produced and selected to achieve the populations that are found normally in the healthy adult brain. The production and initial selection of cortical neurons occur during prenatal life. Many previous studies have identified major features of embryonic development in the central nervous system (CNS) that include neuronal generation within proliferative zones adjacent to the ventricles (the ventricular zone), migration to more superficial locations and subsequent differentiation, and lineage or cohort relationships as revealed by the use of retroviruses. All of these studies have assumed, either explicitly or implicitly, that a negligible amount of programmed cell death (PCD or "apoptosis") occurs during this embryonic phase of cortical development. If, however, PCD is pervasive, then it could have an important role in determining the cells that are present during postnatal life. I will thus test the hypothesis that apoptosis differentially affects ventricular zone neuroblast subpopulations and early postmitotic cortical neurons. I will test the hypothesis in 3 specific Aims over the next 5 years. Aim 1 will identify global anatomical and developmental gradients of apoptotic cells during embryonic cortical development (e.g. dorsal-ventral, lateral-medial, rostral-caudal), relate the gradients to proliferative and postmitotic compartments, and determine how these gradients are affected in Balb/c and cell death mutant mouse CPP32 +/+ (wild-type) as compared to +/-(heterozygote) and -/(homozygote) embryos (Years 1-3). Aim 2 will determine the effect of apoptosis on ventricular zone neuroblast subpopulations in Balb/c and CPP32 +/+ (wild-type) as compared to +/-(heterozygote) and -/- (homozygote) embryos; these studies will focus on identified cells within the ventricular zone (as defined by vzg-1 in situ hybridization). Years 1-5. Aim 3 will determine the effect of apoptosis on embryonic, postmitotic neurons within postmitotic neuroanatomical compartments (defined by png-I in situ hybridization) in Balb/c and CPP32 +/+, +/- and -/- embryos. (Years 1-5). This information will provide new insights into the mechanisms behind developmental neurological disorders, especially those affecting the normal functions of the cerebral cortex.

DESCRIPTION (Adapted from the applicant's the abstract): The cerebral cortex is affected in a range of neuropsychiatric disorders including the disruption of normal cortical development that results in autism, childhood schizophrenia and mental retardation. Understanding normal cerebral cortical development will improve the diagnostic and therapeutic options for these and related disorders. This application will add to our knowledge of how neurons arise during the embryonic formation of the cerebral cortex by exploring a new mechanism: lysophosphatidic acid (LPA) signaling through its first receptor called lysophospholipid A1 receptor (LPA1) or Ventricular zone gene-1 (Vvz-1). LPA is a bioactive lipid that has properties of an extracellular signaling molecule and growth factor in non-neural cell lines. This 5-year ISA/K02 application for salary support will thus test the hypothesis that LPA affects cortical development by influencing neurogenesis through its newly identified cognate receptor, LPA1. The hypothesis will be tested through 4 specific aims. Aim 1 will determine embryonic developmental expression patterns of LPA1 by continuing in situ hybridization analyses, generating specific antisera and using these for immunohistochemical analyses of embryonic cortical development. Aim 2 will determine effects of LPA in primary cortical cultures by employing several distinct primary culture techniques to examine real-time morphological changes, cytoskeletal changes, cell fates and whole-brain organization. These studies will define responses and effects of LPA signaling on embryonic cortical cells in culture. Aim 3 will identify and characterize the function of members of a new lysophospholipid receptor gene family that are also expressed in embryonic cortex, and new genes from this family. Aim 4 will produce and analyze mice lacking LPA1, a necessary approach to assessing biological relevance of LPA signaling in the intact animal, since no specific competitive antagonists are currently available. These aims will provide insights into a new influence on the developing cerebral cortex, and can potentially open new avenues to the study and treatment of a range of neuropsychiatric disorders, through the functioning of this novel group of molecules, the bioactive lysophosopholipids and their cognate receptors.

DESCRIPTION (provided by applicant): Understanding the development of the cerebral cortex and the extracellular signals used by forming neurons has clear relevance to mental and neurological diseases. This proposal continues studies on a new extracellular signaling system that was discovered in prior supported work on the cortex.The simple phospholipid called "lysophosphatidic acid" or "LPA," is now known to function through a set of 3 cognate G-protein coupled receptors LPA1-3 and has influences on the cerebral cortex. Determining the receptor-mediated LPA signaling in the cerebral cortex is the focus of the current proposal. In this competitive renewal, we will test the hypothesis that receptor-mediated LPA signaling acts upon neural precursor cells (NPCs) and young postmitotic neurons to alter cell number, survival and fate in the developing cerebral cortex. These hypotheses will be tested by 3 aims: 1) Determine the role of LPA signaling on NPC cell cycle progression, 2) Determine the role of LPA signaling on NPC and neuronal cell death, and 3) Determine the role of LPA signaling on neuronal differentiation. Loss-of-function analyses utilizing targeted genetic nulls of each LPA receptor will be pursued. Gain-of-function analyses will utilize a new cell culture system in which intact embryonic cortex can be grown ex vivo while maintaining normal anatomical landmarks; receptor over activation by exogenous LPA exposure will be complemented by retroviral-mediated expression of LPA receptors in normal tissue. In addition, rescue experiments utilizing retroviruses on LPA-null tissue will be pursued. It is notable that the small size of these lipid molecules makes them attractive targets for drug design, and results from this proposal could serve as a foundation for new therapies based upon lysophospholipid-mimetic drugs.

DESCRIPTION (provided by applicant): Myelination in the periphery involves the function of Schwann cells. A variety of diseases are associated with abnormal or disrupted myelination, ranging from primary demyelinating neuropathies like Guillain-Barre syndrome, immunological mechanisms like Chronic immune demyelinating polyneuropathy (CIDP), and additionally, implication in the etiology of neuropathic pain. The basic biology of Schwann cells and their role in myelination has been extensively studied. Nevertheless, a new influence on Schwann cell biology identified in our lab could have both basic and potentially therapeutic relevance to diseases associated with peripheral myelination: lysophospholipids, small signaling lipids that include lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), mediating their effects through specific cognate receptors that are expressed on Schwann cells. In this proposal, we will test the hypothesis that lysophospholipid signaling alters Schwann cell biology to influence peripheral myelination. Three aims will test this hypothesis: 1) Determine the role of receptor-mediated lysophospholipid signaling on Schwann cell biology and gene expression; 2) Determine the in vivo effects and receptor selectivity of lysophospholipids on peripheral nerve; 3) Determine the lysophospholipid receptor mechanisms of Schwann celI-DRG myelination in culture. Proof-of-concept studies have demonstrated that lysophospholipid receptors are "druggable" targets, raising the prospect that data from this proposal could help in the development of strategies to treat disorders of peripheral myelination

A growing list of human diseases that result in chromosomal aneuploidy also result in mental and neurological disruption of brain function. Examples of such diseases include the well known Down's Syndrome which is trisomy 21 and is associated with mental retardation, and multiple variegated aneuploidy (MVA) that shows a range of aneuploidies and is associated with microcephaly and mental retardation. Recent studies from our lab have identified neuroprogenitor cells and neurons that are aneuploid in normal brain: these cells have gained or lost entire chromosomes, and are thus aneuploid. What is the function of these normally aneuploid cells, and might they be related to phenotypes observed in systemic aneuploidies? In this proposal we will begin to address the function of normal neural aneuploidy by testing the hypothesis that normal alterations in chromosomal number can alter neural cell fate and survival. Two specific aims will be pursued over the two years of this proposal. Aim 1 will determine the distribution and identity of aneuploid cells in the developing neuraxis and adult brain of mouse and adult human. It will also address the sub-hypothesis that aneuploid cells may involve non-random production or be non-randomly distributed. Spectral Karyotyping (SKY) and single-locus fluorescence in situ hybridization (FISH) will allow assessment of total ploidy within interphase cells. Aim 2 will determine the effects of chromosome loss - "loss of heterozygosity" or LOH - on cell fate using a model system. This aim will begin to assess functional roles for aneuploidy, with a focus on hypoploidy for a single chromosome. Live, monosomic cells can be isolated by use of transgenic mice hemizygous for a GFP marker that has been integrated into chromosomes containing a wild-type allele for a cell differentiation gene (LifR). Hemizygous cells will be cell-sorted to purify GFP-negative cells that have lost their wild-type allele via chromosome loss and assessed for significant effects on cell fate (LifR). This proposal will provide a framework for, and data towards, understanding the functional importance of neural aneuploidy in the nervous system.

A significant cause of infertility is disruption of normal embryo implantation. Molecular influences on this process have not been completely identified. A possible influence on implantation and female fertility might be via a bioactive lysophospholipid called lysophosphatidic acid (LPA). LPA activates G protein-coupled receptors (GPCRs) to exert its signaling effects. We have used homologous recombination to the third LPA receptor (called LPA3), and in preliminary studies, have observed reduced fertility attributed to delayed implantation and aberrant embryo spacing in mice lacking this receptor. Interestingly, the phenotype of LPA3- deficient female mice is remarkably similar to that seen in pregnant rats treated with indomethacin or mice deficient for cytosolic phospholipase A2a (cPLA2a), suggesting,a link between prostaglandins (PGs) and LPA signaling. In this proposal we will test the hypothesis that multiple lysophosphatidic acid (LPA) receptors influence embryo implantation. Three aims will be pursued. Aim 1 will determine roles for LPA signaling during implantation by examining LPA/LPA biosynthetic enzymes, and receptors expressed during implantation, with a focus on LPA3and LPA4. Aim 2 will determine the role of prostaglandins (PGs) on LPA signaling in implantation. Aim 3 will determine the mechanisms through which LPAs signaling interacts with PGs. As a pharmaceutically tractable molecule, lysophospholipid receptors could represent a new target for the therapeutic treatment of infertility. Work from this proposal will lay the groundwork towards realizing this possible therapeutic potential.

[unreadable] DESCRIPTION (Provided by Applicant): Understanding the development of the brain and the extracellular signals used by forming neurons has clear relevance to mental and neurological diseases. This proposal focuses on a new simple phospholipid called "sphingosine 1 -phosphate" or "S1P," that is known to function through a set of 5 cognate G-protein coupled receptors S1P1-5. Four of these receptors are expressed to varying degrees in embryonic brain. Determining the role of receptor-mediated S1P signaling in central nervous system (CNS) development is the focus of the proposed studies. In this proposal, we will test the hypothesis that a sphingolipid, S1P, can alter CNS development through activation of cognate G-protein coupled receptors. This hypothesis will be tested by 3 aims: 1) Determine roles for S1P on embryonic brain development using ex vivo models, 2) Determine roles for S1P on embryonic brain development using in vivo models, and 3) Determine the receptor selectivity of S1P effects in ex vivo and in vivo models. Gain-of-function (GOF) analyses will utilize a) a new cell culture system in which intact embryonic brain or cortex can be grown ex vivo while maintaining normal anatomical landmarks and b) embryonic intraventricular injections of ligands. Both GOF analyses permit receptor over activation by exogenous exposure to S1P or S1P agonists. Receptor selectivity of SIP effects will be determined using S1P receptor mutant & wild type animals combined with GOF experiments. Loss-of-function phenotypes during in vivo embryonic development will also be characterized for relevant S1P receptors. It is notable that the small size of these lipid molecules makes them attractive targets for drug design, and results from this proposal could serve as a foundation for new therapies based upon lysophospholipid-mimetic drugs. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Targeting S1P receptors to prevent hearing loss Cochlear hair cells, the primary functional subunits required for the transduction of vibration into the auditory sensation, are subject to irreversible degeneration and consequent loss of hearing acuity. Hair cell loss occurs as the result of a number of insults, including exposure to prolonged, excessive noise, exposure to toxic drugs, and the inevitable process of senescence. Currently, there are no available therapies to prevent these degenerative processes other than minimizing exposure to damaging conditions. Our previous work has demonstrated that sphingosine 1-phosphate (S1P) signaling plays an important role in the maintenance of hair cell integrity. S1P is a small signaling lipid that specifically agonizes a family of five G protein coupled receptors (S1P1-5). Loss of the receptor known as S1P2 in a knockout mouse model results in progressive degeneration of cochlear and vestibular hair cells, balance defects, and profound deafness. Our data demonstrate that other receptors, including S1P3, are also involved in hair cell survival. In this proposal, we will test the hypothesis that S1P receptor agonism can prevent induced hair cell loss. Two aims will address this hypothesis. Aim 1 will identify all the S1P receptors involved in hair cell survival. This will be approached by careful analysis of knockout mice for each S1P receptor subtype. This information is necessary to pursue the most innovative and risky part of the proposal in Aim 2, which is identification and validation of agonists against the involved receptor subtypes identified in Aim 1 to prevent hair cell death. We have already developed major tools for this proposal including S1P receptor knockouts as well as cell-based assays for screening for S1P receptors. However, the risk will be in identifying proof-of-concept compounds - using the Molecular Libraries Screening Centers Network (MLSCN), or the TSRI-Florida screening facilities - and using them to prevent hearing loss. Importantly, proof-of-concept data derived from these studies could one day lead to medicines that could prevent or limi hearing loss. PUBLIC HEALTH RELEVANCE: Non-congenital hearing loss occurs when auditory hair cells die in the inner ear and can be caused by noise, infection, ototoxic medications or aging. Asymmetric hearing loss or death of vestibular hair cells can also lead to balance defects, such as vertigo. Currently there are no medicinal treatments for reversing hearing loss, and sensory hair cells do not regenerate. This proposal will study the function of a small lipid known as sphingosine 1-phosphate (S1P) in the inner ear. Loss of S1P signaling was recently shown to lead to degeneration of auditory and vestibular hair cells leading to hearing loss and disrupted balance in mice, suggesting S1P may play a protective role. Through a combination of neuroscience, genetics and pharmacology, this study aims to test the hypothesis that induced hearing loss can be prevented by pharmacological activation of specific S1P receptors. The goal of this research is to identify novel chemicals that can prevent hair cell loss that results from ototoxic drugs, or loud noise exposure in mouse models. Such findings may eventually lead to new medicines that could prevent hearing loss.

DESCRIPTION (provided by applicant): Congenital or fetal hydrocephalus (FH) is one of the most common neurological disorders of newborns. It is often associated with head enlargement, accumulating cerebrospinal fluid (CSF) and neurological impairment. There are no curative therapies for hydrocephalus and therapies are generally limited to palliative, surgical approaches that utilize shunts to remove excess CSF. This proposal builds upon a recently reported link between a lipid signaling system and FH: lysophospholipids (LPs) that include LPA (lysophosphatidic acid), S1P (sphingosine 1-phosphate), and other related LPs. Present in blood, LPs could be part of the explanation for the observation that fetal bleeding increases the incidence of FH. This study will develop and characterize an animal model that recapitulates multiple, comorbid histological phenomena in the brain while producing FH. It will also determine the linkage between animal models and human FH by examining human FH CSF and brain tissue. Over a 5-year period, three aims will test the hypothesis that LP receptor mechanisms contribute to the etiology and therapeutic tractability of human FH. Aim 1 will identify LPA chemical forms, including precursors and metabolites, from human hydrocephalic CSF, and assess their ability to induce FH and co-morbid CNS structural changes. Samples obtained through clinical collaborators and brain banks will be assessed towards establishing links between human FH and these animal models. Aim 2 will determine cellular and molecular mechanisms contributing to the development of LPA-induced FH and comorbid changes, and assess their prevention or therapy via targeted agents. This aim will provide new mechanistic insights that have therapeutic potential. Aim 3 will identify new LP mechanisms with therapeutic potential contributing to FH and associated co-morbid changes. This aim could expand the lipid influences on FH and offer distinct therapeutic targets for its treatment. Completion of these aims could open new mechanistic and therapeutic understanding for this common and debilitating disorder.

DESCRIPTION (provided by applicant): In utero exposure to alcohol can have major deleterious effects as documented for fetal alcohol syndrome (FAS) and more recently and broadly as fetal alcohol spectrum disorders (FASD). These disorders are associated with a range of debilitating neurodevelopmental and psychiatric problems after birth. Myriad molecular and cellular defects have been associated with fetal brain exposure to ethanol, which generally lack common, underlying mechanisms. This proposal will examine a newly identified, somatic change in the genomes of central nervous system (CNS) cells produced by fetal ethanol exposure that could help to provide a common mechanistic foundation: mosaic aneuploidies. These cells show somatically produced chromosomal gains and/or losses, constituting an inherent, if surprising element of normal brain organization. Constitutive aneuploidies (where all cells have the same form of aneuploidy) have clear consequences for cellular dysfunction in cancers, and deleterious behavioral consequences as observed in Down Syndrome, suggesting that deviations from the normal mosaic aneuploidy states could contribute to the range of neural deficits seen in FASD. Here, we will test the hypothesis that identifiable changes in neural mosaic aneuploidies represent a common endpoint of prenatal exposure to alcohol. Three aims will be pursued over the next 5 years. Aim 1 will identify effects of fetal alcohol exposure that alter neural progenitor cell (NPC) aneuploidies after embryonic exposure ex vivo. Aim 2 will determine cell fate and functional consequences of alcohol exposure to aneuploid & aneusomic NPC populations during development, and neurons in adult cortical cell populations. Aim 3 will determine neuronal and non-neuronal identities and distributions of specific aneusomies produced by fetal alcohol exposure using a novel in vivo reporter system. Completion of these Aims could provide a new framework for understanding and therapeutically approaching FASD.

DESCRIPTION (provided by applicant): Post-hemorrhagic hydrocephalus (PHH) is a common neurological disorder affecting neonates and infants, characterized by increased head size, cerebrospinal fluid (CSF) accumulation, and CNS disability. Neurosurgical removal of CSF offers palliative treatment, yet affected individuals still suffer from neurological sequelae and a need for continual CSF removal and shunt revisions. No truly disease-modifying therapies are currently available. Our proposal explores a new mechanism in the etiology of PHH, particularly occurring during prenatal or premature life, through the actions of lysophospholipids (LPs). These small, membrane- derived lipids include the glycerophospholipid known as lysophosphatidic acid (LPA) that can be present at high levels in blood or hemorrhagic fluids. LPA activates a family of LPA receptors, and preliminary data demonstrate the involvement of at least one receptor, LPA1, in mediating the actions of hemorrhagic fluids and LPA in promoting PHH in an animal model. This model also recapitulates comorbid changes within the brain that have been associated with prenatal PHH in humans. Three specific aims will be pursued over a 5-year period to test the hypothesis that the initiation and progression of PHH involves LPA signaling that further provides a foundation for developing new, therapeutic approaches. Aim 1. Assess effects of prenatal LPA exposure on neuroanatomical changes associated with PHH. Aim 2. Assess intracranial fluid composition and compartments in the PHH mouse model. Aim 3. Determine LPA-dependent PHH developmental timing and assess therapeutic tractability.

PROJECT SUMMARY/ABSTRACT ! Posterior cortical atrophy (PCA), a variant of sporadic Alzheimer's disease (SAD), displays classic AD amyloid plaque and neurofibrillary tangle pathology but preferentially affects the occipital cortex and visual system. Our recent findings on neuronal genomic mosaicism (NGM) and DNA content variation (DCV) in SAD neurons (Bushman & Kaeser et al., eLIFE) may provide a mechanistic framework for understanding PCA through somatically acquired, pathogenic changes (e.g., genomic amplifications) that occur in some (not all) single cells. These changes include increased DCV, with average gains of over 200 megabase pairs, as well as mosaic APP CNVs in single SAD neurons. The current proposal is designed to understand the role of NGM in PCA. Over the next 3 years, we will test the hypothesis that PCA neurons are enriched in SAD-related forms of NGM including APP CNVs, preferentially increased in occipital over temporal and prefrontal cortex. Two Specific Aims will be pursued. Aim 1 will Assess DNA content variation (DCV) including APP CNVs in neurons from PCA vs. non-PCA SAD and non-diseased brains, and identify affected neuronal subtypes. This aim will provide an understanding of NGM at the level of cortical regions (e.g., occipital and parietal vs. frontal) and layers. Aim 2 will assess CNVs in other known AD genes and will identify novel CNVs showing mosaic alterations amongst occipital lobe PCA neurons. This aim could provide a mechanism for the neuroanatomical regions affected by PCA, via enrichment of one or more AD genes by NGM, and could also identify novel disease-related genes. Completion of both Aims will add a new facet to understanding PCA through NGM, with potential relevance to other SAD variants as well as possible therapeutic targets preferentially affected by NGM.

Abstract Human brain is an exceedingly complex network of spatially organized and functionally connected neurons imbedded in glia. In surpasses the mouse brain by three orders of magnitude in terms of sheer numbers of cells, and likely has a more complex organization structure relevant to human-specific cognitive functions. Defining a complete cell atlas of the human brain, including a full catalog of all cell types (i.e. all parts) and their spatial distribution, is a critical step towards understanding the human cognitive machine. In this project we will greatly expand our previous efforts towards building a complete and spatially resolved cell atlas of the human whole brain, using a combination of three novel technologies for scalable single-cell sequencing and in situ imaging. Novel computational approaches will also be developed for integration and analysis of data at these larger scales. We will systematically apply these methods to MRI-scanned human whole brains to produce reference cell maps for female and male brains, and construct a spatial multi-omic map of the human adult brain.

Project Summary/Abstract Understanding the human brain and its diseases represents an enormous challenge but also an opportunity for bettering human health. Among the many remarkable attributes of the normal brain, its ability to store and retrieve information for a lifetime of learning and memories, remains one of life?s joys and mysteries. Alzheimer?s disease (AD) disrupts these cognitive functions and has enormous personal, familial, and societal costs, compounded by a disturbing absence of disease-modifying therapies despite scores of scientific theories, billions of dollars, decades of research, and hundreds of failed clinical trials. This transformative proposal will meet these challenges through studies on a newly identified molecular mechanism within the brain: neuronal gene recombination (NGR). NGR may alter individual genomes within each neuron by linking neural activity ? both normal and abnormal ? to functional DNA gene sequences within the genomes of post-mitotic neurons, doing so through a process of retro-insertion of RNA sequences to produce genomic cDNAs (gencDNAs). The resulting thousands of gene variants for just a single gene ? the AD gene APP ? offers new explanations for disease progression and the futility of AD therapeutics thus far. Three areas of study will be pursued with a team of proven investigators empowered by world class bioinformatics, Alzheimer?s disease, and neuroscience experts. First, we will define the machinery of NGR in the human brain by identifying involved genes and biochemically characterizing their function. Second, we will formally define NGR relevance to major forms of AD and therapeutics by analyses of a sufficient number of sporadic AD brains, as well as examining relationships to familial AD and Down syndrome towards identifying shared molecular etiologies. These studies will also examine a potential near-term therapy for AD by studying FDA-approved reverse transcriptase inhibitors and their impact on AD endpoints, which would be grounded in a scientific foundation based upon NGR. Third, we will use targeted and unbiased approaches to identify new NGR genes and their relationships to other brain diseases, particularly those involved with sporadic brain disorders beyond AD. These studies are the first to examine NGR, representing a new line of research without prior NIH support, with a scope not amenable to standard NIH mechanisms, towards truly transformational studies of the brain, its diseases, and the enormous challenge of AD.

Project Summary/Abstract Understanding the human brain and its diseases represents an enormous challenge but also an opportunity for improving human health. One of the many remarkable attributes of the normal brain is its ability to store and retrieve information for a lifetime of learning and memories. Alzheimer?s disease (AD) and related dementias (ADRDs) disrupt these cognitive functions and have enormous personal, familial, and societal costs, compounded by a disturbing absence of disease-modifying therapies despite scores of scientific theories, billions of dollars, decades of research, and hundreds of failed clinical trials. This transformative proposal will meet these challenges through studies on a newly identified molecular mechanism within the brain: somatic gene recombination (SGR). SGR may alter individual genomes within each neuron by linking neural activity ? both normal and abnormal ? to functional DNA gene sequences present within the genomes of post-mitotic neurons. We hypothesize that through retro-insertion of RNA sequences, genomic cDNAs (gencDNAs) are formed. We identified thousands of gene variants for just a single gene ? the AD gene, APP ? which offers new explanations for disease progression and the failure of AD therapeutics thus far. This proposal will explore the links between SGR acting on other known or unknown disease loci in ADRDs and test the hypothesis that SGR dysregulation represents a common pathogenic mechanism shared by AD and ADRDs. Three areas of study will be pursued by a team of proven investigators empowered by world class ADRD, neuroscience, and bioinformatics experts. First, we will define the machinery of SGR in the human brain by identifying the involved genes and biochemically characterizing their function. Second, we will use targeted and unbiased approaches to identify new genes undergoing SGR in ADRDs and characterize neuroanatomical expression in relation to the classical hallmarks of the disease. Third, we will explore possible targets to be used as biomarkers and for therapeutics in cell culture and human fluid samples. Importantly, these studies will examine a potential near-term therapy for AD and ADRDs by studying FDA-approved reverse transcriptase inhibitors. These proposed studies are the first to examine SGR in ADRDs and represent a new line of research. The scope of this proposal presents a truly transformational study of the brain, its diseases, and the enormous challenge of understanding and treating ADRDs.

PROJECT SUMMARY/ABSTRACT We have identified somatic gene recombination (SGR) in neurons of the human brain, with particular relevance to sporadic Alzheimer?s disease (SAD) (Nature 563, 639-645 (2018)). This discovery represents a new and functionally significant aspect of genomic mosaicism (eLife;4:e05116 (2015)) that has genuine therapeutic potential through newly identified molecular targets. Indeed, we found that SGR, acting on the AD gene for Amyloid Precursor Protein (APP), produces thousands of distinct forms of APP, some of which are enriched in or unique to AD. The APP gene variations related to AD that were analyzed thus far include copy number variations (CNVs) and at least 11 single-nucleotide variations (SNVs) that were previously reported as pathogenic in familial AD, yet that arose somatically and mosaically in SAD; these variations were absent from non-diseased neurons. SGR utilizes reverse transcriptase (RT) activity on transcribed RNAs that, combined with DNA strand-breaks and APP gene transcription, produce double-stranded DNA that is retro-inserted back into the genome to form ?genomic cDNAs? (gencDNAs). These published data contribute to the scientific foundation on which the current proposal will build, to test the hypothesis that altered SGR, involving brain- specific reverse transcriptases, functionally contributes to AD and affects multiple genes, providing novel targets for AD therapies. Postmortem IRB-approved and de-identified brain samples from validated AD donors of both sexes will be compared to non-diseased controls, while IACUC-approved animal experiments will model SGR and its AD-relevant endpoints. Three Aims will be pursued over 5 years. Aim 1 will define the molecular neurobiology of APP gencDNA diversity and identify new SGR genes enhanced in AD brains. Aim 2 will determine expression and function of SGR genes in AD brain and model systems. Aim 3 will identify genes responsible for RT SGR activity within normal and AD brains. This proposal will thus open new vistas into AD via novel SGR mechanisms and will identify new therapeutic targets for the treatment of AD.