Mitochondrial diseases caused by mutations in mitochondrial DNA (mtDNA) are a significant source of morbidity and mortality, yet only supportive care is available to affected patients. This lack of effective therapies can be partly attributed to the lack of faithful animal models for mitochondrial diseases caused by mutations in mtDNA. Two critical obstacles to the availability of mouse models of these diseases are 1) the lack of mouse mtDNA mutations homologous to those found in human disease, and 2) the lack of a method to efficiently generate such mutations. Therefore, the long-term GOALS of the proposed studies are 1) to overcome these obstacles in order to enable routine modeling of human diseases caused by mtDNA mutations, and 2) to provide mouse models of mitochondrial disease to the research community. In this application, we propose to leverage expertise developed at the University of South Alabama (mutagenesis of mouse mtDNA) and the Children?s Hospital of Philadelphia (transmitochondrial mouse models) towards generating models of human diseases caused by mutations in mitochondrially-encoded tRNA genes. Using the tools and techniques developed in the previous funding cycle and in preliminary studies, we will 1) generate an arrayed library of mouse clones carrying mutations in mtDNA, 2) use it to perform a targeted screening for mutations in tRNALeuUUR and in tRNALys, and 3) use these mutations as well as tRNA mutations isolated by us previously for the generation and characterization of transmitochondrial animals. It is anticipated that, if successful, the proposed studies will deliver a powerful impetus to mitochondrial research in general and to research on mitochondrial disease in particular by providing the mitochondrial research community with (1) mouse model(s) for mitochondrial disease caused by mutations in tRNA genes, (2) an arrayed library of mouse clones suitable for targeted screening for mtDNA mutations of interest as well as tools and protocols for generating and screening such libraries, and (3) cell line(s) carrying mouse homolog(s) of human pathogenic mtDNA mutations. In this competitive revision, we request funds to procure equipment that will dramatically facilitate the generation and storage of an arrayed library of mouse cell lines containing mutations in mtDNA. While the generation of a collection of mutant mammalian cell lines is not without precedent, the generation of an arrayed library of cell lines with mutations in mtDNA has not been attempted before. In the previous funding cycle, our attempts to generate and screen a large representative library of mouse mtDNA mutants were hampered by low throughput of cryopreservation and sequencing protocols. The equipment that will be procured with the requested funds will introduce semi-automation into our attempts at cryopreservation of the clones, and will enable storage of clones in an 8x12 array format, which is amenable to medium- and high- throughput manipulation and screening.

Public Health Relevance

In this competitive revision of our R01 OD010944 application (funded on 01/01/2016), we request funds to purchase equipment for the arraying and liquid nitrogen storage of clones with mutations in mtDNA, which are the subject of the parent grant. This revision broadens the scope of the original award and requests support for ongoing research activities to facilitate the generation of animal models of mitochondrial disease. Purchased equipment will be installed in the Gene Delivery Core and therefore will be accessible (and beneficial) to all researchers who need to generate and store a large number of mammalian cell line clones (e.g., CRISPR- Cas9 generated libraries of ES cell or iPS cell mutants).

Agency
National Institute of Health (NIH)
Institute
Office of The Director, National Institutes of Health (OD)
Type
Research Project (R01)
Project #
3R01OD010944-06S1
Application #
9324583
Study Section
Scientific and Technical Review Board on Biomedical and Behavioral Research Facilities (STOD)
Program Officer
Mccullough, Willie
Project Start
2010-06-01
Project End
2017-11-30
Budget Start
2017-04-01
Budget End
2017-11-30
Support Year
6
Fiscal Year
2017
Total Cost
$75,000
Indirect Cost
Name
University of South Alabama
Department
Biology
Type
Schools of Medicine
DUNS #
172750234
City
Mobile
State
AL
Country
United States
Zip Code
36688
Khozhukhar, Natalya; Spadafora, Domenico; Rodriguez, Yelitza et al. (2018) Elimination of Mitochondrial DNA from Mammalian Cells. Curr Protoc Cell Biol 78:20.11.1-20.11.14
Pei, Liming; Wallace, Douglas C (2018) Mitochondrial Etiology of Neuropsychiatric Disorders. Biol Psychiatry 83:722-730
Ferreira, C R; Goorden, S M I; Soldatos, A et al. (2018) Deoxysphingolipid precursors indicate abnormal sphingolipid metabolism in individuals with primary and secondary disturbances of serine availability. Mol Genet Metab 124:204-209
Wallace, Douglas C (2017) A Mitochondrial Etiology of Neuropsychiatric Disorders. JAMA Psychiatry 74:863-864
Angelin, Alessia; Gil-de-Gómez, Luis; Dahiya, Satinder et al. (2017) Foxp3 Reprograms T Cell Metabolism to Function in Low-Glucose, High-Lactate Environments. Cell Metab 25:1282-1293.e7
Barel, Ortal; Malicdan, May Christine V; Ben-Zeev, Bruria et al. (2017) Deleterious variants in TRAK1 disrupt mitochondrial movement and cause fatal encephalopathy. Brain 140:568-581
Kandel, Judith; Picard, Martin; Wallace, Douglas C et al. (2017) Mitochondrial DNA 3243A>G heteroplasmy is associated with changes in cytoskeletal protein expression and cell mechanics. J R Soc Interface 14:
Zand, Katayoun; Pham, Ted D A; Li, Jinfeng et al. (2017) Resistive flow sensing of vital mitochondria with nanoelectrodes. Mitochondrion 37:8-16
Balczon, Ron; Morrow, K Adam; Zhou, Chun et al. (2017) Pseudomonas aeruginosa infection liberates transmissible, cytotoxic prion amyloids. FASEB J 31:2785-2796
Shokolenko, Inna N; Alexeyev, Mikhail F (2017) Mitochondrial transcription in mammalian cells. Front Biosci (Landmark Ed) 22:835-853

Showing the most recent 10 out of 55 publications