Mike Grotewiel

Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila

Age-related behavioral declines are common manifestations of aging in animals. Negative geotaxis, an innate escape response during which flies ascend the wall of a cylinder after being tapped to its bottom, is one of the behaviors that senesces in Drosophila. Many laboratories, including ours, have used a variety of negative geotaxis assays based on the performance of single flies. To circumvent limitations of single-fly assays, we developed a new method for assessing negative geotaxis called rapid iterative negative geotaxis (RING). In RING assays, digital photography is used to document negative geotaxis in multiple groups of animals simultaneously. We show that performance in RING assays is not influenced by the density of flies being tested, the time of day, or repeated testing. We used the RING assay to demonstrate that negative geotaxis declines with the age of animals as previously shown in single fly studies and that senescence of negative geotaxis is sensitive to genetic background. Finally, we used RING assays to show that long-lived Indy and chico mutants exhibit delayed senescence of negative geotaxis. Our results demonstrate that RING is a powerful method for assessing negative geotaxis that should facilitate the search for manipulations that influence behavioral aging in Drosophila.

Oxidative damage and age-related functional declines.

Most organisms experience progressive declines in physiological function as they age. Since this senescence of function is thought to underlie the decrease in quality of life in addition to the increase in susceptibility to disease and death associated with aging, identifying the mechanisms involved would be highly beneficial. One of the leading mechanistic theories for aging is the oxidative damage hypothesis. A number of studies in a variety of species support a strong link between oxidative damage and life span determination. The role of oxidative damage in functional senescence has also been investigated, albeit not as comprehensively. Here, we review these investigations. Several studies show that the age-related loss of a number of functions is associated with an accrual of oxidative damage in the tissues mediating those functions. Additionally, treatments that increase the accumulation of oxidative damage with age frequently exacerbate functional losses. Moreover, treatments that reduce the accumulation of oxidative damage often attenuate or delay the loss of function associated with aging. These data provide the foundation for a link between oxidative damage and functional senescence, thereby supporting the oxidative damage hypothesis of aging within the context of age-related functional decline.

Reduced mitochondrial SOD displays mortality characteristics reminiscent of natural aging.

Manganese superoxide dismutase (MnSOD or SOD2) is a key mitochondrial enzymatic antioxidant. Arguably the most striking phenotype associated with complete loss of SOD2 in flies and mice is shortened life span. To further explore the role of SOD2 in protecting animals from aging and age-associated pathology, we generated a unique collection of Drosophila mutants that progressively reduce SOD2 expression and function. Mitochondrial aconitase activity was substantially reduced in the Sod2 mutants, suggesting that SOD2 normally ensures the functional capacity of mitochondria. Flies with severe reductions in SOD2 expression exhibited accelerated senescence of olfactory behavior as well as precocious neurodegeneration and DNA strand breakage in neurons. Furthermore, life span was progressively shortened and age-dependent mortality was increased in conjunction with reduced SOD2 expression, while initial mortality and developmental viability were unaffected. Interestingly, life span and age-dependent mortality varied exponentially with SOD2 activity, indicating that there might normally be a surplus of this enzyme for protecting animals from premature death. Our data support a model in which disruption of the protective effects of SOD2 on mitochondria manifests as profound changes in behavioral and demographic aging as well as exacerbated age-related pathology in the nervous system.

Drosophila as a model for age-related impairment in locomotor and other behaviors

Aging is a multifaceted phenomenon that occurs in most species including humans and the fruit fly, Drosophila melanogaster. One of the most fundamental features of aging is the progressive decline in functional capacity that occurs with age (i.e. functional senescence). Age-related declines in function undermine many aspects of normal youthful physiology including behavior. Age-related behavioral declines are quite telling because they presumably reflect primary functional defects in the nervous system or musculature. Consequently, a more detailed understanding of behavioral declines that occur with age, including mechanisms that impinge on them, could ultimately lead to improved treatment or diagnosis of age-related defects in physiological processes that depend on normal function of the nervous system or musculature. Such advances in diagnosis or treatment would translate into tremendous gains in quality of life for elderly populations. In this article, we review progress using Drosophila to better understand age-related behavioral declines with a focus on age-related locomotor impairment.

Smith-Magenis Syndrome Results in Disruption of CLOCK Gene Transcription and Reveals an Integral Role for RAI1 in the Maintenance of Circadian Rhythmicity

Haploinsufficiency of RAI1 results in Smith-Magenis syndrome (SMS), a disorder characterized by intellectual disability, multiple congenital anomalies, obesity, neurobehavioral abnormalities, and a disrupted circadian sleep-wake pattern. An inverted melatonin rhythm (i.e., melatonin peaks during the day instead of at night) and associated sleep-phase disturbances in individuals with SMS, as well as a short-period circadian rhythm in mice with a chromosomal deletion of Rai1, support SMS as a circadian-rhythm-dysfunction disorder. However, the molecular cause of the circadian defect in SMS has not been described. The circadian oscillator temporally orchestrates metabolism, physiology, and behavior largely through transcriptional modulation. Data support RAI1 as a transcriptional regulator, but the genes it might regulate are largely unknown. Investigation into the role that RAI1 plays in the regulation of gene transcription and circadian maintenance revealed that RAI1 regulates the transcription of circadian locomotor output cycles kaput (CLOCK), a key component of the mammalian circadian oscillator that transcriptionally regulates many critical circadian genes. Data further show that haploinsufficiency of RAI1 and Rai1 in SMS fibroblasts and the mouse hypothalamus, respectively, results in the transcriptional dysregulation of the circadian clock and causes altered expression and regulation of multiple circadian genes, including PER2, PER3, CRY1, BMAL1, and others. These data suggest that heterozygous mutation of RAI1 and Rai1 leads to a disrupted circadian rhythm and thus results in an abnormal sleep-wake cycle, which can contribute to an abnormal feeding pattern and dependent cognitive performance. Finally, we conclude that RAI1 is a positive transcriptional regulator of CLOCK, pinpointing a novel and important role for this gene in the circadian oscillator.

Genetic and environmental factors impact age-related impairment of negative geotaxis in Drosophila by altering age-dependent climbing speed

Age-related locomotor impairment in humans is important clinically because it is associated with several co-morbidities and increased risk of death. One of the hallmarks of age-related locomotor impairment in humans is a decrease in walking speed with age. Genetically tractable model organisms such as Drosophila are essential for delineating mechanisms underlying age-related locomotor impairment and age-related decreases in locomotor speed. Negative geotaxis, the ability of flies to move vertically when startled, is a common measure of locomotor behavior that declines with age in Drosophila. Toward further developing Drosophila as a model for age-related locomotor impairment, we investigated whether negative geotaxis reflects climbing or a combination of climbing and other behaviors such as flying and jumping. Additionally, we investigated whether locomotor speed in negative geotaxis assays declines with age in flies as found for walking speed in humans. We find that the vast majority of flies climb during negative geotaxis assays and that removal of hind legs, but not wings, impairs the behavior. We also find that climbing speed decreases with age in four wild type genetic backgrounds, in flies housed at different temperatures, and in control and long-lived flies harboring a mutation in OR83b. The decreases in climbing speed correlate with the age-related impairments in the distance climbed. These studies establish negative geotaxis in Drosophila as a climbing behavior that declines with age due to a decrease in climbing speed. Age-related decreases in locomotor speed are common attributes of locomotor senescence in flies and humans.

Multi-species data integration and gene ranking enrich significant results in an alcoholism genome-wide association study.

BackgroundA variety of species and experimental designs have been used to study genetic influences on alcohol dependence, ethanol response, and related traits. Integration of these heterogeneous data can be used to produce a ranked target gene list for additional investigation.ResultsIn this study, we performed a unique multi-species evidence-based data integration using three microarray experiments in mice or humans that generated an initial alcohol dependence (AD) related genes list, human linkage and association results, and gene sets implicated in C. elegans and Drosophila. We then used permutation and false discovery rate (FDR) analyses on the genome-wide association studies (GWAS) dataset from the Collaborative Study on the Genetics of Alcoholism (COGA) to evaluate the ranking results and weighting matrices. We found one weighting score matrix could increase FDR based q-values for a list of 47 genes with a score greater than 2. Our follow up functional enrichment tests revealed these genes were primarily involved in brain responses to ethanol and neural adaptations occurring with alcoholism.ConclusionsThese results, along with our experimental validation of specific genes in mice, C. elegans and Drosophila, suggest that a cross-species evidence-based approach is useful to identify candidate genes contributing to alcoholism.

Chloride intracellular channels modulate acute ethanol behaviors in Drosophila, Caenorhabditis elegans and mice

Identifying genes that influence behavioral responses to alcohol is critical for understanding the molecular basis of alcoholism and ultimately developing therapeutic interventions for the disease. Using an integrated approach that combined the power of the Drosophila, Caenorhabditis elegans and mouse model systems with bioinformatics analyses, we established a novel, conserved role for chloride intracellular channels (CLICs) in alcohol‐related behavior. CLIC proteins might have several biochemical functions including intracellular chloride channel activity, modulation of transforming growth factor (TGF)‐β signaling, and regulation of ryanodine receptors and A‐kinase anchoring proteins. We initially identified vertebrate Clic4 as a candidate ethanol‐responsive gene via bioinformatic analysis of data from published microarray studies of mouse and human ethanol‐related genes. We confirmed that Clic4 expression was increased by ethanol treatment in mouse prefrontal cortex and also uncovered a correlation between basal expression of Clic4 in prefrontal cortex and the locomotor activating and sedating properties of ethanol across the BXD mouse genetic reference panel. Furthermore, we found that disruption of the sole Clic Drosophila orthologue significantly blunted sensitivity to alcohol in flies, that mutations in two C. elegans Clic orthologues, exc‐4 and exl‐1, altered behavioral responses to acute ethanol in worms and that viral‐mediated overexpression of Clic4 in mouse brain decreased the sedating properties of ethanol. Together, our studies demonstrate key roles for Clic genes in behavioral responses to acute alcohol in Drosophila, C. elegans and mice.

Dietary restriction alters demographic but not behavioral aging in Drosophila.

Dietary restriction extends lifespan substantially in numerous species including Drosophila. However, it is unclear whether dietary restriction in flies impacts age‐related functional declines in conjunction with its effects on lifespan. Here, we address this issue by assessing the effect of dietary restriction on lifespan and behavioral senescence in two wild‐type strains, in our standard white laboratory stock, and in short‐lived flies with reduced expression of superoxide dismutase 2. As expected, dietary restriction extended lifespan in all of these strains. The effect of dietary restriction on lifespan varied with genetic background, ranging from 40 to 90% extension of median lifespan in the seven strains tested. Interestingly, despite its robust positive effects on lifespan, dietary restriction had no substantive effects on senescence of behavior in any of the strains in our studies. Our results suggest that dietary restriction does not have a global impact on aging in Drosophila and support the hypothesis that lifespan and behavioral senescence are not driven by identical mechanisms.

Sod2 knockdown in the musculature has whole-organism consequences in Drosophila

Oxidative damage to cell macromolecules by reactive oxygen species is associated with numerous diseases and aging. In Drosophila, RNAi-mediated silencing of the mitochondrial antioxidant manganese superoxide dismutase (SOD2) throughout the body dramatically reduces life span, accelerates senescence of locomotor function, and enhances sensitivity to applied oxidative stress. Here, we show that Sod2 knockdown in the musculature alone is sufficient to cause the shortened life span and accelerated locomotor declines observed with knockdown of Sod2 throughout the body, indicating that Sod2 deficiency in muscle is central to these phenotypes. Knockdown of Sod2 in the muscle also increased caspase activity (a marker for apoptosis) and caused a mitochondrial pathology characterized by swollen mitochondria, decreased mitochondrial content, and reduced ATP levels. These findings indicate that Sod2 plays a crucial role in the musculature in Drosophila and that the consequences of SOD2 loss in this tissue extend to the viability of the organism as a whole.