The long-term goal of the proposed research is to study the mechanisms responsible for age-related degenerative processes of the human brain. The understanding of these mechanisms may help to find ways to slow the corresponding processes thus moving the onset of deterioration outside the normal human lifespan. We propose to explore if accumulation of somatic mutations in mtDNA of critical cell types in the brain is one of the causative factors in the age-related deterioration of the brain. Two hypotheses are proposed. First, mutations in mtDNA could work as the primary cause of the dysfunction of certain brain areas by disrupting cellular metabolism, facilitating cell death, increasing the generation of reactive oxygen radicals, and possibly other mechanisms. Second, the presence of mutated mtDNA may render aged cells sensitive to various biochemical insults associated with specific late-onset neurodegenerative diseases. These hypotheses are supported by our preliminary finding that a majority of individual pigmented neurons in the substantia nigra in the old but not in the young brain accumulates very high levels of clonally expanded mtDNA deletions. The observed levels of deletions in pigmented neurons are above the physiological threshold and thus are highly likely to interfere with cellular function as well as with the ability of the cell to respond to the various stresses. Key to testing of these hypotheses is the analysis of the distribution of mtDNA mutations and physiological states of the cells in various areas of the brain at the single cell level, which represents the core of the proposed research.
The specific aims of the application are: (1) To develop and optimize the arsenal of methods necessary for precise quantification and characterization of mtDNA mutations in single cells of the brain. These methods will include laser capture microdissection for single cell isolation, amplification of full length mitochondrial genomes from single cells, single cell competitive PCR, and single cell limiting dilution PCR. (2) To identify brain areas and cell types in which mtDNA mutations are most likely to play a causative role in the aging process. This will be done by measuring mutation load in individual cells of substantia nigra, cortex and putamen. These areas are known to be rich in mtDNA deletions and are associated with brain functions that decline with age, and are affected in the major late onset neurodegenerative diseases. (3) To test the hypothesis that clonal expansions of mtDNA mutations in individual cells contribute to mitochondrial defects, neural dysfunction and degeneration in normal aging and late-onset neurodegenerative diseases. This will be done by comparing the mutational load of cells that stained positive for various markers of mitochondrial dysfunction, oxidative stress and cell degeneration to non-staining control cells. We will also study the distribution the mutations as a function of age and the severity of the disease.
|Safdar, Adeel; Annis, Sofia; Kraytsberg, Yevgenya et al. (2016) Amelioration of premature aging in mtDNA mutator mouse by exercise: the interplay of oxidative stress, PGC-1?, p53, and DNA damage. A hypothesis. Curr Opin Genet Dev 38:127-132|
|Safdar, Adeel; Khrapko, Konstantin; Flynn, James M et al. (2016) Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice. Skelet Muscle 6:7|
|Greaves, Laura C; Nooteboom, Marco; Elson, Joanna L et al. (2014) Clonal expansion of early to mid-life mitochondrial DNA point mutations drives mitochondrial dysfunction during human ageing. PLoS Genet 10:e1004620|
|Campbell, Graham R; Kraytsberg, Yevgenya; Krishnan, Kim J et al. (2012) Clonally expanded mitochondrial DNA deletions within the choroid plexus in multiple sclerosis. Acta Neuropathol 124:209-20|
|Khrapko, Konstantin (2011) The timing of mitochondrial DNA mutations in aging. Nat Genet 43:726-7|
|Guo, Xinhong; Popadin, Konstantin Yu; Markuzon, Natalya et al. (2010) Repeats, longevity and the sources of mtDNA deletions: evidence from 'deletional spectra'. Trends Genet 26:340-3|
|Guo, Xinhong; Kudryavtseva, Elena; Bodyak, Natalya et al. (2010) Mitochondrial DNA deletions in mice in men: substantia nigra is much less affected in the mouse. Biochim Biophys Acta 1797:1159-62|
|Nicholas, A; de Magalhaes, J P; Kraytsberg, Y et al. (2010) Age-related gene-specific changes of A-to-I mRNA editing in the human brain. Mech Ageing Dev 131:445-7|
|Zsurka, Gabor; Kudina, Tatiana; Peeva, Viktoriya et al. (2010) Distinct patterns of mitochondrial genome diversity in bonobos (Pan paniscus) and humans. BMC Evol Biol 10:270|
|Greaves, Laura C; Beadle, Nina E; Taylor, Geoffrey A et al. (2009) Quantification of mitochondrial DNA mutation load. Aging Cell 8:566-72|
Showing the most recent 10 out of 33 publications