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1.  Assessing Basal and Acute Autophagic Responses in the Adult Drosophila Nervous System: The Impact of Gender, Genetics and Diet on Endogenous Pathway Profiles 
PLoS ONE  2016;11(10):e0164239.
The autophagy pathway is critical for the long-term homeostasis of cells and adult organisms and is often activated during periods of stress. Reduced pathway efficacy plays a central role in several progressive neurological disorders that are associated with the accumulation of cytotoxic peptides and protein aggregates. Previous studies have shown that genetic and transgenic alterations to the autophagy pathway impacts longevity and neural aggregate profiles of adult Drosophila. In this study, we have identified methods to measure the acute in vivo induction of the autophagy pathway in the adult fly CNS. Our findings indicate that the genotype, age, and gender of adult flies can influence pathway responses. Further, we demonstrate that middle-aged male flies exposed to intermittent fasting (IF) had improved neuronal autophagic profiles. IF-treated flies also had lower neural aggregate profiles, maintained more youthful behaviors and longer lifespans, when compared to ad libitum controls. In summary, we present methodology to detect dynamic in vivo changes that occur to the autophagic profiles in the adult Drosophila CNS and that a novel IF-treatment protocol improves pathway response in the aging nervous system.
doi:10.1371/journal.pone.0164239
PMCID: PMC5053599  PMID: 27711219
2.  Chronic Voluntary Ethanol Consumption Induces Favorable Ceramide Profiles in Selectively Bred Alcohol-Preferring (P) Rats 
PLoS ONE  2015;10(9):e0139012.
Heavy alcohol consumption has detrimental neurologic effects, inducing widespread neuronal loss in both fetuses and adults. One proposed mechanism of ethanol-induced cell loss with sufficient exposure is an elevation in concentrations of bioactive lipids that mediate apoptosis, including the membrane sphingolipid metabolites ceramide and sphingosine. While these naturally-occurring lipids serve as important modulators of normal neuronal development, elevated levels resulting from various extracellular insults have been implicated in pathological apoptosis of neurons and oligodendrocytes in several neuroinflammatory and neurodegenerative disorders. Prior work has shown that acute administration of ethanol to developing mice increases levels of ceramide in multiple brain regions, hypothesized to be a mediator of fetal alcohol-induced neuronal loss. Elevated ceramide levels have also been implicated in ethanol-mediated neurodegeneration in adult animals and humans. Here, we determined the effect of chronic voluntary ethanol consumption on lipid profiles in brain and peripheral tissues from adult alcohol-preferring (P) rats to further examine alterations in lipid composition as a potential contributor to ethanol-induced cellular damage. P rats were exposed for 13 weeks to a 20% ethanol intermittent-access drinking paradigm (45 ethanol sessions total) or were given access only to water (control). Following the final session, tissues were collected for subsequent chromatographic analysis of lipid content and enzymatic gene expression. Contrary to expectations, ethanol-exposed rats displayed substantial reductions in concentrations of ceramides in forebrain and heart relative to non-exposed controls, and modest but significant decreases in liver cholesterol. qRT-PCR analysis showed a reduction in the expression of sphingolipid delta(4)-desaturase (Degs2), an enzyme involved in de novo ceramide synthesis. These findings indicate that ethanol intake levels achieved by alcohol-preferring P rats as a result of chronic voluntary exposure may have favorable vs. detrimental effects on lipid profiles in this genetic line, consistent with data supporting beneficial cardioprotective and neuroprotective effects of moderate ethanol consumption.
doi:10.1371/journal.pone.0139012
PMCID: PMC4583526  PMID: 26405804
3.  Disruption of Sphingolipid Metabolism Elicits Apoptosis-Associated Reproductive Defects in Drosophila 
Developmental biology  2007;309(2):329-341.
Sphingolipid signaling is thought to regulate apoptosis via mechanisms that are dependent on the concentration of ceramide relative to that of sphingosine-1-phosphate (S1P). This study reports defects in reproductive structures and function that are associated with enhanced apoptosis in Drosophila Sply05091 mutants that lack functional S1P lyase and thereby accumulate sphingolipid long chain base metabolites. Analyses of reproductive structures in these adult mutants unmasked multiple abnormalities, including supernumerary spermathecae, degenerative ovaries, and severely reduced testes. TUNEL assessment revealed increased cell death in mutant egg chambers at most oogenic stages and in affected mutant testes. These reproductive abnormalities and elevated gonadal apoptosis were also observed, to varying degrees, in other mutants affecting sphingolipid metabolism. Importantly, the reproductive defects seen in the Sply05091 mutants were ameliorated both by a second site mutation in the lace gene that restores long chain base levels towards normal and by genetic disruption of the proapoptotic genes reaper, hid and grim. These data thus provide the first evidence in Drosophila that accumulated sphingolipids trigger elevated levels of apoptosis via the modulation of known signaling pathways.
doi:10.1016/j.ydbio.2007.07.021
PMCID: PMC2094363  PMID: 17706961
Sphingolipid; Ceramide; S1P; Apoptosis; Cell death; Drosophila; Oogenesis; Spermatogenesis; Reproduction; Germ cells
4.  Aging and Autophagic Function Influences the Progressive Decline of Adult Drosophila Behaviors 
PLoS ONE  2015;10(7):e0132768.
Multiple neurological disorders are characterized by the abnormal accumulation of protein aggregates and the progressive impairment of complex behaviors. Our Drosophila studies demonstrate that middle-aged wild-type flies (WT, ~4-weeks) exhibit a marked accumulation of neural aggregates that is commensurate with the decline of the autophagy pathway. However, enhancing autophagy via neuronal over-expression of Atg8a (Atg8a-OE) reduces the age-dependent accumulation of aggregates. Here we assess basal locomotor activity profiles for single- and group-housed male and female WT flies and observed that only modest behavioral changes occurred by 4-weeks of age, with the noted exception of group-housed male flies. Male flies in same-sex social groups exhibit a progressive increase in nighttime activity. Infrared videos show aged group-housed males (4-weeks) are engaged in extensive bouts of courtship during periods of darkness, which is partly repressed during lighted conditions. Together, these nighttime courtship behaviors were nearly absent in young WT flies and aged Atg8a-OE flies. Previous studies have indicated a regulatory role for olfaction in male courtship partner choice. Coincidently, the mRNA expression profiles of several olfactory genes decline with age in WT flies; however, they are maintained in age-matched Atg8a-OE flies. Together, these results suggest that middle-aged male flies develop impairments in olfaction, which could contribute to the dysregulation of courtship behaviors during dark time periods. Combined, our results demonstrate that as Drosophila age, they develop early behavior defects that are coordinate with protein aggregate accumulation in the nervous system. In addition, the nighttime activity behavior is preserved when neuronal autophagy is maintained (Atg8a-OE flies). Thus, environmental or genetic factors that modify autophagic capacity could have a positive impact on neuronal aging and complex behaviors.
doi:10.1371/journal.pone.0132768
PMCID: PMC4504520  PMID: 26182057
5.  Identification of Sphingolipid Metabolites That Induce Obesity via Misregulation of Appetite, Caloric Intake and Fat Storage in Drosophila 
PLoS Genetics  2013;9(12):e1003970.
Obesity is defined by excessive lipid accumulation. However, the active mechanistic roles that lipids play in its progression are not understood. Accumulation of ceramide, the metabolic hub of sphingolipid metabolism, has been associated with metabolic syndrome and obesity in humans and model systems. Here, we use Drosophila genetic manipulations to cause accumulation or depletion of ceramide and sphingosine-1-phosphate (S1P) intermediates. Sphingolipidomic profiles were characterized across mutants for various sphingolipid metabolic genes using liquid chromatography electrospray ionization tandem mass spectroscopy. Biochemical assays and microscopy were used to assess classic hallmarks of obesity including elevated fat stores, increased body weight, resistance to starvation induced death, increased adiposity, and fat cell hypertrophy. Multiple behavioral assays were used to assess appetite, caloric intake, meal size and meal frequency. Additionally, we utilized DNA microarrays to profile differential gene expression between these flies, which mapped to changes in lipid metabolic pathways. Our results show that accumulation of ceramides is sufficient to induce obesity phenotypes by two distinct mechanisms: 1) Dihydroceramide (C14:0) and ceramide diene (C14:2) accumulation lowered fat store mobilization by reducing adipokinetic hormone- producing cell functionality and 2) Modulating the S1P: ceramide (C14:1) ratio suppressed postprandial satiety via the hindgut-specific neuropeptide like receptor dNepYr, resulting in caloric intake-dependent obesity.
Author Summary
Obesity is characterized by excessive weight gain that increases one's risk for pathologies such as Type II diabetes and heart disease. It is well-known that a high calorie diet rich in saturated fats contributes to excessive weight gain. However, the role that saturated fats play in this process goes far beyond simple storage in fat tissue. Saturated fats are essential building blocks for the bioactive lipid ceramide. Accumulation of ceramide has recently been associated with obesity. However, it is not known whether its accumulation plays an active role in the induction of obesity. Here, we utilized genetic manipulation in Drosophila to accumulate and deplete a variety of ceramide species and other related lipids. Our results showed that modulation of ceramide and related lipids is sufficient to induce obesity through two distinct mechanisms: a caloric intake-dependent mechanism works through suppression of neuropeptide Y satiety signaling, while a caloric intake-independent mechanism works through regulation of hormone producing cells that regulate fat storage. These data implicate ceramides in actively promoting obesity by increasing caloric intake and fat storage.
doi:10.1371/journal.pgen.1003970
PMCID: PMC3854795  PMID: 24339790
6.  Survival Response to Increased Ceramide Involves Metabolic Adaptation through Novel Regulators of Glycolysis and Lipolysis 
PLoS Genetics  2013;9(6):e1003556.
The sphingolipid ceramide elicits several stress responses, however, organisms survive despite increased ceramide but how they do so is poorly understood. We demonstrate here that the AKT/FOXO pathway regulates survival in increased ceramide environment by metabolic adaptation involving changes in glycolysis and lipolysis through novel downstream targets. We show that ceramide kinase mutants accumulate ceramide and this leads to reduction in energy levels due to compromised oxidative phosphorylation. Mutants show increased activation of Akt and a consequent decrease in FOXO levels. These changes lead to enhanced glycolysis by upregulating the activity of phosphoglyceromutase, enolase, pyruvate kinase, and lactate dehydrogenase to provide energy. A second major consequence of AKT/FOXO reprogramming in the mutants is the increased mobilization of lipid from the gut through novel lipase targets, CG8093 and CG6277 for energy contribution. Ubiquitous reduction of these targets by knockdown experiments results in semi or total lethality of the mutants, demonstrating the importance of activating them. The efficiency of these adaptive mechanisms decreases with age and leads to reduction in adult life span of the mutants. In particular, mutants develop cardiac dysfunction with age, likely reflecting the high energy requirement of a well-functioning heart. The lipases also regulate physiological triacylglycerol homeostasis and are important for energy metabolism since midgut specific reduction of them in wild type flies results in increased sensitivity to starvation and accumulation of triglycerides leading to cardiac defects. The central findings of increased AKT activation, decreased FOXO level and activation of phosphoglyceromutase and pyruvate kinase are also observed in mice heterozygous for ceramide transfer protein suggesting a conserved role of this pathway in mammals. These data reveal novel glycolytic and non-autonomous lipolytic pathways in response to increased ceramide for sustenance of high energy demanding organ functions like the heart.
Author Summary
Ceramide belongs to an important class of lipids called sphingolipids. An increase in ceramide levels elicits several stress responses. We have used Drosophila mutated in ceramide kinase, an enzyme involved in metabolizing ceramide to understand how an organism adapts to stress imposed by ceramide. We find that in the mutants, increased ceramide decreases energy production via mitochondrial oxidative phosphorylation. The mutants compensate for decreased energy by upregulating glycolysis, a central pathway of intermediary metabolism. Another important mechanism that is activated in these ceramide kinase mutants is lipolysis, which is involved in breakdown of triglycerides for energy contribution. The mutants show increased hydrolysis of triglycerides in the gut, probably to provide sufficient energy for optimal performance of the heart, a high energy demanding organ. The upregulation of these pathways in the mutant result from increased activation of AKT and decreased FOXO. The modulation of glycolysis and lipolysis by AKT/FOXO is mediated by novel targets identified in this study. Failure of these adaptive mechanisms in aging flies lead to triglyceride accumulation, cardiac abnormalities, and ultimately a decrease in the lifespan of the mutant animals. The mechanisms identified here could have clinical implications in diseases associated with ceramide such as diabetes and obesity.
doi:10.1371/journal.pgen.1003556
PMCID: PMC3688504  PMID: 23818862
7.  p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects 
Autophagy  2011;7(6):572-583.
Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.
doi:10.4161/auto.7.6.14943
PMCID: PMC3127048  PMID: 21325881
p62; Ref(2)P; insoluble ubiquitinated proteins; aggregates; neural degeneration; Alzheimer disease; aging; macroautophagy
8.  In Vitro and In Vivo Antagonism of a G Protein-Coupled Receptor (S1P3) with a Novel Blocking Monoclonal Antibody 
PLoS ONE  2012;7(4):e35129.
Background
S1P3 is a lipid-activated G protein-couple receptor (GPCR) that has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. Currently, there are no available high-affinity, subtype-selective drug compounds that can block activation of S1P3. We have developed a monoclonal antibody (7H9) that specifically recognizes S1P3 and acts as a functional antagonist.
Methodology/Principal Findings
Specific binding of 7H9 was demonstrated by immunocytochemistry using cells that over-express individual members of the S1P receptor family. We show, in vitro, that 7H9 can inhibit the activation of S1P3-mediated cellular processes, including arrestin translocation, receptor internalization, adenylate cyclase inhibiton, and calcium mobilization. We also demonstrate that 7H9 blocks activation of S1P3 in vivo, 1) by preventing lethality due to systemic inflammation, and 2) by altering the progression of breast tumor xenografts.
Conclusions/Significance
We have developed the first-reported monoclonal antibody that selectively recognizes a lipid-activated GPCR and blocks functional activity. In addition to serving as a lead drug compound for the treatment of sepsis and breast cancer, it also provides proof of concept for the generation of novel GPCR-specific therapeutic antibodies.
doi:10.1371/journal.pone.0035129
PMCID: PMC3320623  PMID: 22496900

Results 1-8 (8)