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Genetic Dissection of Aging in Drosophila

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    1. Genetic Dissection of Aging in Drosophila Goals Identify the genes involved in aging and longevity Use knowledge of these genes to determine the physiological systems important in aging and longevity How? Mutagenesis What phenotype to use?

    2. Logistical problems using Life span as a phenotype in a mutagenesis Longest lived member of a population is less likely to breed

    3. How do we measure aging? Demographic measures of aging Physiological measures of aging

    5. Aging: populations versus individuals Measurement of life expectancy in a population. Measurement of life expectancy in individuals. Is chronological age the same as physiological age?

    6. Life span of fruit flies at three temperatures

    11. Validity of use of beta-gal as a marker of gene expression during adult life. Little change in somatic cell populations in the adult fly. Protein synthesis is preserved in many cells of the adult fly throughout life. Rate of degradation of beta-gal is on the order of several hours.

    14. Environmental manipulation of life span

    15. Life span of fruit flies at three temperatures

    18. Changing life span changes the chronological timing of 1085 gene expression

    19. 1085 gene expression scales to life span

    20. Genetic manipulation of life span

    26. Biomarkers of aging

    28. Wg expression scales with lifespan: using ambient temperature

    29. Hyperkinetic and Shaker are single gene mutations that accelerate the rate of aging. Sex-linked neurological mutants. Die earlier than normal animals. Slope of mortality curve indicates an acceleration of the aging process. Oxygen consumption is increased over normal. Physical activity is increased over normal.

    31. Wg expression scales with lifespan: using Shaker mutation

    32. Wg expression scales with lifespan: using Hyperkinetic mutation

    33. Loss of SOD function shortens life span

    37. drop dead (drd) Mutations in drd lead to adult onset neurodegeneration and early death. (Hotta and Benzer, Proc Natl Acad Sci U S A. 1970 Nov;67(3):1156-63) Drop dead is X-linked recessive. Mosaic analysis suggests it is non cell autonomous The age of death appears stochastic and is effected by ambient temperature.

    45. Idealized biomarker expression scales to life span

    47. Coupling biomarker activity to the expression of a lethal toxin enriches for long-lived mutant flies delayed in accumulating the lethal toxin. Biomarker threshold detection by using a deadly toxin

    48. To obtain spatial control of hUCP2 expression we adopted the widely used, bipartite yeast UAS-Gal4 expression system first developed by Brand and Perrimon. Here is a schematic illustrating how the system works. The first step is to generate transgenic flies carrying the UAS-hUCP2 construct. This construct allows the transcription of the hUCP2 only in the presence of the transcriptional activator GAL 4 which binds to the UAS motifs and turns on transcription. So the transgene remains inactive until the fly harboring this construct is crossed to the driver lines producing the Gal4 protein in either a ubiquitous or tissue specific manner depending upon the promoter. To obtain spatial control of hUCP2 expression we adopted the widely used, bipartite yeast UAS-Gal4 expression system first developed by Brand and Perrimon. Here is a schematic illustrating how the system works. The first step is to generate transgenic flies carrying the UAS-hUCP2 construct. This construct allows the transcription of the hUCP2 only in the presence of the transcriptional activator GAL 4 which binds to the UAS motifs and turns on transcription. So the transgene remains inactive until the fly harboring this construct is crossed to the driver lines producing the Gal4 protein in either a ubiquitous or tissue specific manner depending upon the promoter.

    49. DJ651 and DJ634 are two age-dependent GAL4 enhancer-trap lines that demonstrate rapid up-regulation after eclosion which scaled to life span. DJ651 and DJ634 are two age-dependent GAL4 enhancer-trap lines that demonstrate rapid up-regulation after eclosion which scaled to life span.

    50. Is the toxin induced death age-dependent? Examine how interventions that alter life span and rate of aging affect rate of toxin induced death?

    51. Temperature is an environmental intervention know to effect life span in poikilotherms. This increase is similar but slightly less than what is seen when normal flies are cultured at these temperatures Temperature is an environmental intervention know to effect life span in poikilotherms. This increase is similar but slightly less than what is seen when normal flies are cultured at these temperatures

    52. Reproductive status influences life span in insects. A decrease in reproduction, particularly in females, is know to extend life span significantly. Virgin females can live up to 100% longer than mated females. Male virgins see a less significant increase in life span around 10-20% which was recapitulated by the system. At this point the system has recapitulated all three environmental manipulation that are known to extend life span in normal adult flies the system also shows a similar magnitude as that of normal flies. At this point the system can be used to screen for drugs that effect life span. Reproductive status influences life span in insects. A decrease in reproduction, particularly in females, is know to extend life span significantly. Virgin females can live up to 100% longer than mated females. Male virgins see a less significant increase in life span around 10-20% which was recapitulated by the system. At this point the system has recapitulated all three environmental manipulation that are known to extend life span in normal adult flies the system also shows a similar magnitude as that of normal flies. At this point the system can be used to screen for drugs that effect life span.

    53. Calorie restriction is an environmental intervention that increase life span in flies as well as various other species. Low Calorie Food contains half the calories of normal food. The percentage of life span increase seen with DTT flies is similar in magnitude to that of normal flies around 50%. Calorie restriction is an environmental intervention that increase life span in flies as well as various other species. Low Calorie Food contains half the calories of normal food. The percentage of life span increase seen with DTT flies is similar in magnitude to that of normal flies around 50%.

    55. Females heterozygous for the hypomorphic allele show a greater increase that females carrying the null mutationFemales heterozygous for the hypomorphic allele show a greater increase that females carrying the null mutation

    57. Screen for genes or drugs that change biomarker/gene trajectory

    58. Lipoic Acid

    60. Resveratrol

    62. Logistical problems with the tetanus toxin mutagenesis screen not much time to select long-lived candidates, before they are also overcome by the toxin and die or are infertile.

    63. Temporal control over the Toxin Tetanus Toxin-not conditional Inducible/conditional expression Cyanide produced by Linamarase/Linamarin

    66. To obtain spatial control of hUCP2 expression we adopted the widely used, bipartite yeast UAS-Gal4 expression system first developed by Brand and Perrimon. Here is a schematic illustrating how the system works. The first step is to generate transgenic flies carrying the UAS-hUCP2 construct. This construct allows the transcription of the hUCP2 only in the presence of the transcriptional activator GAL 4 which binds to the UAS motifs and turns on transcription. So the transgene remains inactive until the fly harboring this construct is crossed to the driver lines producing the Gal4 protein in either a ubiquitous or tissue specific manner depending upon the promoter. To obtain spatial control of hUCP2 expression we adopted the widely used, bipartite yeast UAS-Gal4 expression system first developed by Brand and Perrimon. Here is a schematic illustrating how the system works. The first step is to generate transgenic flies carrying the UAS-hUCP2 construct. This construct allows the transcription of the hUCP2 only in the presence of the transcriptional activator GAL 4 which binds to the UAS motifs and turns on transcription. So the transgene remains inactive until the fly harboring this construct is crossed to the driver lines producing the Gal4 protein in either a ubiquitous or tissue specific manner depending upon the promoter.

    70. To obtain spatial control of hUCP2 expression we adopted the widely used, bipartite yeast UAS-Gal4 expression system first developed by Brand and Perrimon. Here is a schematic illustrating how the system works. The first step is to generate transgenic flies carrying the UAS-hUCP2 construct. This construct allows the transcription of the hUCP2 only in the presence of the transcriptional activator GAL 4 which binds to the UAS motifs and turns on transcription. So the transgene remains inactive until the fly harboring this construct is crossed to the driver lines producing the Gal4 protein in either a ubiquitous or tissue specific manner depending upon the promoter. To obtain spatial control of hUCP2 expression we adopted the widely used, bipartite yeast UAS-Gal4 expression system first developed by Brand and Perrimon. Here is a schematic illustrating how the system works. The first step is to generate transgenic flies carrying the UAS-hUCP2 construct. This construct allows the transcription of the hUCP2 only in the presence of the transcriptional activator GAL 4 which binds to the UAS motifs and turns on transcription. So the transgene remains inactive until the fly harboring this construct is crossed to the driver lines producing the Gal4 protein in either a ubiquitous or tissue specific manner depending upon the promoter.

    71. DJ651-Lin1.4

    72. Summary Gene expression during adult life continues to be well regulated Gene expression may serve as a biomarker of aging Age-dependent gene expression can be used to screen for drugs and genes that affect life span

    73. Acknowledgments National Institute on Aging National Science Foundation Ellison Medical Foundation American Federation for Aging Research The Glenn Foundation for Medical Research Donaghue Foundation

    74. Feynman on aging

    75. Researchers at the Mayo Clinic reported that a 30-year study has revealed that optimistic people live 19 percent longer than pessimists. ("I knew it!" said the optimists. "Just goes to show," muttered the pessimists.)