Disturbed phospholipid metabolism in serine biosynthesis defects revealed by metabolomic profiling.

Serine biosynthesis defects are autosomal recessive metabolic disorders resulting from the deficiency of any of the three enzymes involved in de novo serine biosynthesis, specifically phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). In this study, we performed metabolomic profiling on 4 children with serine biosynthesis defects; 3 with PGDH deficiency and 1 with PSAT deficiency. The evaluations were performed at baseline and with serine and glycine supplementation. Metabolomic profiling performed at baseline showed low phospholipid species, including glycerophosphocholine, glycerophosphoethanolamine, and sphingomyelin. All children had low serine and glycine as expected. Low glycerophosphocholine compounds were found in 4 children, low glycerophosphoethanolamine compounds in 3 children, and low sphingomyelin species in 2 children. Metabolic profiling with serine and glycine supplementation showed normalization of most of the low phospholipid compounds in the 4 children. Phospholipids are the major component of plasma and intracellular membranes, and phosphatidylcholine is the most abundant phospholipid of all mammalian cell types and subcellular organelles. Phosphatidylcholine is of particular importance for the nervous system, where it is essential for neuronal differentiation. The observed low phosphatidylcholine species in children with serine biosynthesis defects that improved after serine supplementation, supports the role of serine as a significant precursor for phosphatidylcholine. The vital role that phosphatidylcholine has during neuronal differentiation and the pronounced neurological manifestations in serine biosynthesis defects suggest that phosphatidylcholine deficiency occurring secondary to serine deficiency may have a significant contribution to the development of the neurological manifestations in individuals with serine biosynthesis defects.

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.

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

BACKGROUND: A 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. RESULTS: In 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. CONCLUSIONS: These 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.

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.

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.

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.

Distinct genetic influences on locomotor senescence in Drosophila revealed by a series of metrical analyses.

Decline of locomotor function is a prominent feature of aging in most species. We previously described a series of metrical analyses for quantifying functional declines associated with aging. Here, we have applied these metrics to elucidate differences in the nature of locomotor senescence between Drosophila cohorts from four different genetic backgrounds (Canton-S, Oregon-R, Samarkand and Lausanne-S) and two long-lived mutant strains, Indy(206) and chico(1). These analyses demonstrate that senescence of locomotor function in Oregon-R and Canton-S proceeds more rapidly than in Samarkand and Lausanne-S flies. Our analyses also show that while chico(1) and Indy mutants both exhibit attenuated locomotor declines, the impact of chico loss of function on locomotor senescence is substantially greater. Our studies illustrate how important differences in functional senescence characteristics can be unveiled by in-depth analysis on functional senescence data sets.

A proposed set of descriptors for functional senescence data.

Declines in function are common manifestations of aging in many phyla. Because functional senescence is thought to drive the increasing risk of death with age, understanding functional senescence is important for understanding aging. Experimental investigation of functional senescence requires one to quantitative and compare age-dependent declines in function between cohorts. Such quantitation and comparison is often difficult due to complexities in functional senescence data sets. Here, we discuss issues related to describing and contrasting age-related declines in function. We parameterized functional senescence data in simple ways to generate descriptors for (1) rate of functional decline, (2) time to onset of functional decline, and (3) total function. To illustrate how these descriptors can be used, we analyzed a hypothetical data set and one of our previously published data sets. We conclude that no one descriptor sufficiently characterizes functional senescence. Useful distinctions between functional senescence in different cohorts can be made, however, when multiple descriptors are used in an integrated fashion [corrected]

Functional senescence in Drosophila melanogaster.

The fruit fly Drosophila melanogaster is one of the principal model organisms used for studying the biology of aging. Flies are well suited for such studies for a number of reasons. Flies develop to adulthood quickly, have a relatively short life span, and are inexpensive to house. Most of the fly genome has been sequenced, powerful genetic tools are available to manipulate it, and most fly genes have obvious homologues in mammals. While the majority of aging studies in flies have focused on regulation of life span, the fly is emerging as a powerful model system for investigating the biology that underlies age-related functional decline. Key to the use of flies in this way is the striking number of parallels between functional senescence in Drosophila and humans. Here, we review age-related functional declines in Drosophila, human correlates of these age-related declines, and common mechanisms that influence longevity and specific aspects of functional senescence in flies.

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.

Dissociation between functional senescence and oxidative stress resistance in Drosophila.

Many studies strongly suggest a causal link between oxidative stress and determination of life span. The relationship between oxidative stress and age-related functional declines, however, is less clear. Additionally, the full spectrum of functional declines associated with aging has not been systematically evaluated in the fruit fly, Drosophila melanogaster, one of the leading models for aging research. Toward a more comprehensive assessment of functional senescence in Drosophila, we evaluated a series of behaviors in control flies of increasing ages. Our studies reveal a novel age-dependent functional decline in the olfactory system and confirm previous reports of age-related locomotor defects in flies. Behavioral responses to electric shock and light are maintained in aged flies. Thus, some sensory systems senesce during the first several weeks of life while others do not. Interestingly, the age-dependent functional declines in olfactory and locomotor systems are indistinguishable in control flies and methuselah, a mutant with enhanced resistance to oxidative stress and increased life span. Our results indicate that enhanced resistance to oxidative stress and extension of life span do not necessarily confer protection from age-related functional declines.

Behavioral responses to odorants in drosophila require nervous system expression of the beta integrin gene myospheroid.

Integrins are cell adhesion molecules that mediate numerous developmental processes in addition to a variety of acute physiological events. Two reports implicate a Drosophila beta integrin, betaPS, in olfactory behavior. To further investigate the role of integrins in Drosophila olfaction, we used Gal4-driven expression of RNA interference (RNAi) transgenes to knock down expression of myospheroid (mys), the gene that encodes betaPS. Expression of mys-RNAi transgenes in the wing reduced betaPS immunostaining and produced morphological defects associated with loss-of-function mutations in mys, demonstrating that this strategy knocked down mys function. Expression of mys-RNAi transgenes in the antennae, antennal lobes, and mushroom bodies via two Gal4 lines, H24 and MT14, disrupted olfactory behavior but did not alter locomotor abilities or central nervous system structure. Olfactory behavior was normal in flies that expressed mys-RNAi transgenes via other Gal4 lines that specifically targeted the antennae, the projection neurons, the mushroom bodies, bitter and sweet gustatory neurons, or Pox neuro neurons. Our studies confirm that mys is important for the development or function of the Drosophila olfactory system. Additionally, our studies demonstrate that mys is required for normal behavioral responses to both aversive and attractive odorants. Our results are consistent with a model in which betaPS mediates events within the antennal lobes that influence odorant sensitivity.