Michael D. Koob - US grants

nucleic acid repetitive sequence

DESCRIPTION (Investigator's abstract): Spinocerebellar ataxia type 8 (SCA8) is caused by a CTG expansion in an untranslated, endogenous antisense RNA that overlaps the Kelch-like 1 (KLHL1) gene. The KLHL1 promoter and open-reading frame are conserved in mouse, and a KLHL1.antisense transcript (KLHL1AS) is present in mouse as well. We have performed initial characterization of the KLHL1AS and KLHL1 genes in both man and mouse, but we are at this point left with some fundamental questions regarding these genes: 1) What is the normal function of the evolutionarily conserved KLHL1-antisense RNA?, 2) What role does the KLHL1 protein play in the neurons in which it is expressed?, and 3) How does the CTG expansion affect the KLHL1AS RNA and KLHL1 protein, and can these effects explain the neurodegeneration seen in SCA8 patients? The data we have obtained to date have allowed us to make informed hypotheses concerning these questions and to design some in vivo experimental systems to test, refine, and if necessary reformulate these hypotheses. The transcriptional organization of the KLHL1 mRNA and the KLHL1AS transcript suggests that KLHL1AS is most likely a regulator of KLHL1 expression. Based on the homology of KLHL1 to other neuron-specific kelch-like proteins and on our preliminary characterization of this protein, we speculate that KLHL1 may play a role in organizing the actin fibers that help to generate and maintain axons and dendrites. We cannot predict a priori how the SCA8 CTG expansion may affect the KLHL1/KLHL1AS gene system. Both of these genes, however, are specifically expressed in the cerebellum, and so these transcripts are likely candidates for mediating the pathogenic effect of this expansion either directly or through altered antisense interactions. We will perform multiple, iterative modifications of a BAC clone encoding the human KLHLAS gene and the first two exons of KLHL1 in E. coli using homologous recombination. We will then introduce the modified BAC clones into tissue culture cel Is to directly determine how KLHL1 and KLHL1AS interact and measure what effect these interactions have on KLHL1 expression levels. Replacing the CTG repeat in the last KLHL1AS exon with expanded repeats in these clones will also allow us to test how the SCA8 expansion affects KLHL1AS and KLHL1, and how it alters the interactions between these genes. Although we will continue to characterize the structure, protein interactions and cellular and subcellular localization of the KIHI1 protein, we will directly test the role of this protein in normal neuronal development and function by generating a KLHL1 gene knockout in mouse. We have designed our knockout strategy in a way that will allow us to both 1) study the effects of disrupting the KLHL1 gene in a tissue-specific and temporal-specific manner, and 2) determine the effect that altered levels of KLHL1AS transcription has on KLHL1 expression and function.

DESCRIPTION (provided by applicant): Mouse models of human genetic diseases have been critical for confirming that particular mutations cause disease, for obtaining a detailed molecular understanding of the pathophysiology of these diseases, and for developing and testing potential therapies. Technical limitations, however, currently prevent us from generating accurate mouse models of human diseases caused by mutations in our mitochondria! DNA. Although several groups have shown that they can generate mice that contain mitochondrial genomes isolated from mouse tissue culture cells, the technology has not yet been developed that allows us to introduce specific nucleotide changes into mouse mitochondrial genomes. We are proposing to overcome this technical limitation. We have cloned the complete mouse mitochondrial genome stably in E. coli and have shown that we can introduce essentially any desired mutation into these clones. We have also shown that we can introduce these engineered mitochondrial genomes into the mitochondria of yeast cells devoid of their own mtDNA and that these mouse mtDNA genomes are replicated accurately in these cells. We now propose to use these transgenomic yeast mitochondria as vectors for transferring the engineered mtDNA genomes back into the mitochondrial networks of mouse tissue culture cells, which will then be used to generate mice with modified mitochondrial genomes. Our specific aims for this project are to 1) develop and optimize methods for using transgenomic yeast mitochondria as mitochondrial delivery vectors that will efficiently fuse to mouse mitochondrial networks, and to 2) determine how to best screen or select for mouse cells that have been transformed with modified mitochondrial genomes.

DESCRIPTION (provided by applicant): A wide array of inherited human diseases are caused by mutations in our mitochondrial DNA (mtDNA) genome and in mitochondrial genes encoded in the nuclear genome, but there are currently no effective therapies for these clinically devastating diseases. We are able to engineer yeast mtDNA and, moreover, we have developed technology that allows us for the first time to engineer mammalian mitochondrial genomes and reintroduce these genomes into mouse embryos. We propose to use this technology to develop a gene therapy for Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disease caused by defects in frataxin, a nucleus-encoded mitochondrial protein. We will focus our initial efforts on correcting the molecular deficits associated with the complete loss of frataxin in yeast. This well characterized model of FRDA will enable us to systematically assess both general and protein-specific features required to efficiently express a fully functional form of frataxin from the mitochondrial genome. We will use the information and reagents developed in this initial phase of the project to engineer a set of mouse mtDNA genomes suitable for correcting deficits in a mouse model of FRDA. We will evaluate the efficiency with which these genomes compensate for the loss of the mouse nuclear frataxin gene by packaging them in mitochondria and injecting them into single cell embryos of FRDA knockout mice. Because this gene knockout mutation leads to loss of mitochondrial function and so is embryonic lethal, we can readily assay the functionality of the mitochondrial frataxin genes by their ability to either partially rescue (i.e., generate viable embryonic cells) or fully rescue this phenotype (i.e., generate viable mice). This experimental system will therefore allow us to optimize both our mitochondrial transfer technology and our mitochondrial frataxin gene constructs. Once we have completed the work described in this application we will be in an excellent position to develop a gene therapy approach for mouse models of FRDA and to work towards adapting these therapies to treating FRDA and other mitochondrial diseases in humans. PUBLIC HEALTH RELEVANCE: A wide array of inherited human diseases are caused by mutations in our mitochondrial DNA (mtDNA) genome and in mitochondrial genes encoded in the nuclear genome, but there are currently no effective therapies for these clinically devastating diseases. The experiments described in this proposal will give us both greater molecular insights into one of these diseases (FRDA) and will allow us to develop mtDNA engineering and transfer technologies that will serve as indispensable tools for developing therapies to treat these diseases.

Project Summary Our long-term goal is to understand the biological mechanisms underlying GWAS hits in LOAD and other tauopathies. The objective of our current proposal is to develop mouse models in which the entire 190 Kb human MAPT gene precisely replaces the complete 157 Kb mouse Mapt gene, and to use these models to compare the molecular endophenotypes of tau in young and old mice expressing H2 or H1 MAPT haplotypes. Our rationale is that the non-coding variants that define the H1 and H2 haplotypes are present in MAPT but not Mapt, necessitating the incorporation of the full MAPT gene. In addition, because we have found that the location of MAPT in the mouse genome affects its expression, we will preserve the basic structural configuration of Chr17q21.31 in which MAPT resides, and maintain its relationship relative to other genes in the gene cluster that mice and humans share. These studies will be significant because they will constitute the first in vivo bioassays of non-coding polymorphisms, paving the way for future research on GWAS hits in not only LOAD, but also other chronic medical disorders. Our overarching hypothesis is that differences in the progression and pattern of tau mRNA and protein expression underlie the variations in risk associated with H2 and H1 MAPT haplotypes. We have in-hand the first MAPT Gene-Replacement (GR) mouse line, in which the 190kb human H2 MAPT precisely replaces the 157 Kb Mapt locus, and will use the same methodology to create a precisely matched line in which the H1 MAPT replaces Mapt. We will measure and compare mRNA, protein and post-translational modifications of tau in young and old GR1 and GR2 mice. Upon completion of these studies, we will have created and characterized two lines of mice in which Mapt is precisely replaced by H1 or H2 variants of MAPT. We predict that the distinct polymorphisms in H1 and H2 MAPT will suffice to alter the molecular endophenotype of tau in GR1 relative to GR2 mice, and that these differences will increase with age.

This proposal is aimed at developing two gene-replacement mouse models ? one that expresses a wildtype human Noxa protein and a second that expresses an apoptosis-deficient version of human Noxa, by replacing the murine NOXA gene and promoter sequence with the corresponding region of human NOXA using a bacterial artificial chromosome (BAC). Human (h) Noxa, a 54-residue protein, was initially identified as a ?pro-apoptotic? member of the Bcl-2 family, that could interact with pro-survival family member Mcl-1 via its BH3 domain to promote apoptosis. However, it was discovered later that hNoxa also promotes growth and proliferation in human hematopoietic lineage cells. This growth-promoting function, evident in both normal and malignant hematopoietic cells, has been attributed to Noxa?s ability to reprogram metabolism. In T cell and other leukemias, hNoxa is phosphorylated on a single serine (S13) by a glucose-dependent kinase. This eliminates its ability to bind Mcl-1 and promote apoptosis but enhances its ability to support metabolic reprogramming that increases glucose and glutamine uptake for growth and proliferation. Recent data point to a similar growth-promoting metabolic role for Noxa in primary human T cells following antigenic stimulation, leading to their rapid expansion and differentiation. Taken together, our studies point to a central and unique role for hNoxa in T cell development, expansion and differentiation. Its dual opposing functions make hNoxa a potential target for cancer therapy and for immunotherapy. Mouse models will be essential for in vivo validation and for further defining Noxa?s role in apoptosis and metabolism. Mouse (m)Noxa, differs from hNoxa, both structurally and in its regulation, has not been shown to promote growth or reprogram metabolism. In the first aim, we will generate hNOXA gene-replacement (GR) mice by replacing the entire mouse NOXA gene and promoter region with the corresponding region of hNOXA. An ES cell line will first be generated in which a portion of the mouse genome harboring the NOXA gene is deleted and replaced by a site-specific recombination target cassette. Next, the syntenic NOXA genomic sequence from the human genome will be inserted into the cassette in the prepared ES cell line. The second aim will be directed at determining whether hNoxa is accurately expressed and functional in the WT hNOXA mouse. Initial testing will focus on Noxa expression in CD8 T lymphocytes following in vitro activation, and on the impact of hNoxa expression on the generation, function and maintenance of effector and memory CD8+T cells in vivo. The DA hNOXA GR mouse, expressing an apoptosis- deficient BH3 domain mutant of Noxa, will be generated after determining that WT hNOXA promoter elements are functional and express hNoxa. If successfully generated, these mice could help validate hNoxa?s role in T cell metabolism during an immune response, offer insights into hNoxa?s role in leukemogenesis, and serve as pre-clinical models for testing therapeutic strategies to activate its pro-apoptotic function.

We are responding to NOT-AG-18-049 ?Collaborative Studies on AD/ADRD? by establishing a collaborative effort to significantly expand the modeling capacity of the MODEL-AD Center. MODEL-AD was established by the NIA to create, rigorously characterize and ensure the rapid distribution of the next generation of animal models of Late Onset AD. Critical barriers to progress in making these next generation models are technique limitations that have put restrictions on the size of the human genomic context incorporated into each of the new modified alleles in the MODEL-AD mouse lines. Our group at the University of Minnesota (UMN) has developed Gene Replacement (GR) technologies that allow us to routinely replace mouse genes with their full human orthologs up to several hundred kb in size. We used this technology to generate a matched set of Microtubule Associated Protein Tau Gene- Replacement (MAPT-GR) lines of mice in which we replaced the full mouse Mapt genomic coding and regulatory region (156,547bp) with full human MAPT genomic sequences (190,081bp). We have confirmed that mice homozygous for this MAPT-GR allele express human tau at endogenous levels, and that all expected splice variants are found in the appropriate tissues and in ratios expected for the fully functional human MAPT gene. This model set now includes two wt control lines (H1 or H2 MAPT haplotype) and a growing number of experimental lines that precisely match the H1 wt control line except for the pathogenic variant that we specifically introduce into that haplotype. The first of these lines are currently being further characterized by MODEL-AD and are now available to the AD research community without restriction (JAX). Our specific aims for this collaboration are to: 1. Generate Gene-Replacement (GR) sets of mouse lines in which genes involved in the etiology of AD have been replaced by their full human homologs. We are proposing to develop 10 model sets for this collaboration (>20 total lines). 2. Characterize matched sets of GR lines using the established MODEL-AD methods and distribute without restriction. The most translationally relevant alleles will be incorporated into the current MODEL-AD ?base model?. These GR lines will allow us and other AD researchers to evaluate the molecular impact of pathogenic mutations and risk variants within the context of the full human gene sequence in which they occur in patients. These mouse lines will contain all potential human therapeutic targets for each gene, ranging from the full genomic DNA sequences to all RNA transcription and protein products that they encode. Because the genomic sequences of these matched sets will differ only at sequences specifically changed in each line, any significant molecular differences between these lines can confidently be attributed to the risk variant in the experimental lines, and any therapeutic agents found to effectively correct these dysfunctions could be expected to have direct therapeutic value to patients.