Study of sphingolipid-mediated signaling and metabolism during aging

Sphingolipids are a class of bioactive signaling molecules that regulate key cellular processes including cell growth, senescence and apoptosis, and have been implicated in age-related neurodegeneration. Previous studies have reported elevated ceramide levels in the brain of old rodents, but a systematic investigation of the impact of age on brain sphingolipid metabolism was still lacking. In the present study we quantified 17 key sphingolipid species in the hippocampus of young (3 months), middle-aged (12 months) and old (21 months) male and female mice. Lipids were extracted and quantified by liquid chromatography/mass spectrometry; transcription of enzymes involved in sphingolipid biosynthesis was evaluated by qPCR. Age-dependent changes of multiple sphingolipid species - including ceramide (d18:1/18:0), sphingomyelin (d34:1), hexosylceramide (d18:1/16:0), ceramide (d18:1/24:0) - were found in mice of both sexes. Moreover, sex-dependent changes were seen with hexosylceramide (d18:1/18:0), ceramide (d18:1/22:0), sphingomyelin (d36:1) and sphingomyelin (d42:1). Importantly, an age-dependent accumulation of sphingolipids containing nervonic acid (24:1) was observed in 21 month-old male (p = 0.04) and female mice (p < 0.001). Consistent with this increase, transcription of the nervonic acid-synthesizing enzyme, stearoyl-CoA desaturase (Scd1 and Scd2), was upregulated in 21 month-old female mice (Scd1 p = 0.006; Scd2 p = 0.009); a similar trend was observed in males (Scd1 p = 0.07). In conclusion, the results suggest that aging is associated with profound sex-dependent and -independent changes in hippocampal sphingolipid profile. The results also highlight the need to examine the contribution of sphingolipids, and particularly of those containing nervonic acid, in normal and pathological brain aging. Nevertheless, also the circulating ceramids are altered in persons affected by age-dependent pathologies such as metabolic syndrome, mild cognitive impairment and Alzheimer’s disease, but the potential impact of age and gender on plasma ceramide trajectories in healthy subjects has not been systematically examined. In this study we quantified a panel of circulating ceramides and dihydroceramides in a cohort of 164 healthy subjects (84 female, 80 male; 19-80 years of age). The results show that plasma ceramide levels are significantly lower (p < 0.05) in pre-menopausal women (aged 20-54 years) compared to age-matched men (aged 19-54 years). This difference disappears after menopause, such that plasma ceramide levels in post-menopausal women (aged 47-78 years) are statistically identical to those measured in aged-matched men (aged 55-80 years). In women of all ages, but not in men, circulating levels of ceramide (d18:1/24:1) were negatively correlated with plasma estradiol levels. Finally, in vitro experiment showed that incubation with estradiol (10 nM, 24 h) lowers ceramide levels in the human MCF7 breast cancer cell line. Together the results suggest the existence of gender- and age-dependent alterations in circulating ceramide concentrations, which are dependent on estradiol. In addition to my previous study on rodent model, the present work introduces menopause and fluctuating estradiol levels as new variables to keep into account in the study of aging. Aging is the main risk factor for the development of neurodegenerative diseases such as Alzheimer's disease but also chronic diseases such as metabolic syndrome. Indeed, it is becoming increasingly evident that cellular and biochemical alterations observed in metabolic syndrome like, among others, alterations in lipid mediators, may represent a pathological bridge between age-related neurological disorders and metabolic syndrome. Sphingolipids have been implicated in the pathogenesis of metabolic dysfunction, but physiological signals regulating their formation and deactivations in hypothalamus are unknown. Hypothalamus is an especially important node in central and peripheral regulation of feeding behavior. We studied the effect of high-fat diet (HFD) or food deprivation (FD) on sphingolipid levels and on the expression of enzymes involved in sphingolipid metabolism in the hypothalamus. To study the effect of HFD, mice were divided into two groups: standard diet (2.66 kcal/g) and HFD (5.24 kcal/g) and killed at different time points (1-3-7-14-28 days). To study the effect of fasting, male mice were subjected to 4 feeding conditions: 1) free feeding (FF); 2) 12h food deprivation (FD); 3) 1h refeeding after FD; 4) 6h refeeding after FD. Hypothalamic sphingolipids were extracted and quantified by LC-MS/MS. Transcription of enzymes involved in sphingolipid biosynthesis was evaluated by qPCR. After 1 day and 14 days, mice exposed to a HFD showed lower levels of ceramide (d18:1/24:0), its precursor dihydroceramide (d18:0/24:0), and ceramide (d18:1/24:1) compared to mice fed standard diet. Significant decrease in sphingosine-1-phosphate (SO-1-P) was also observed after 7 and 14 days of HFD. Relative to FF, fasting: 1) decreased SO-1-P levels; 2) increased sphingosine, the precursor of sphingosine-1-phosphate; 3) reduced the levels of dihydroceramide (d18:0/18:0), a product of de novo ceramide biosynthesis; 4) down-regulated transcription of sphingosine kinases (SphK) and ceramide synthase 1 (CerS1). Our results suggest that hypothalamic levels of SO-1-P, its precursor sphingosine and enzymes involved in their metabolism (SphK) are influenced by feeding status. Feeding also regulates the de novo synthesis of sphingolipid, suggesting additional roles for these lipids in the control of energy balance. Finally, a single intracerebroventricular injection of an acid ceramidase inhibitor, ARN14974, which has been shown to imbalance the ceramide/sphingosine-1-phosphate rheostat, was able to alter some parameters of feeding behavior such as meal size. The aim of my studies was to explore the complexity of sphingolipid metabolism and understand the role of sphingolipids as lipid-derived mediators of cell signaling in physiological or altered conditions in rodent model and in human. Aging and feeding states are strongly correlated since aging has been associated with development of obesity and metabolic disorders, which depend, in turn, on altered feeding status; on the other hand, hyperphagia or fasting may have respectively detrimental or beneficial effect on aging and longevity. Furthermore, since a variety of studies have been published on the relevance of dimorphism in the development of age-related disorders, I pursued my research keeping into account sex-related differences. Brain areas and plasma have been chosen as target tissues for my studies. My research has been focused on specific cerebral areas involved respectively in aging and cognition (hippocampus) and feeding control and energy balance (hypothalamus). To investigate the role of ceramides in human, I had access to plasma from healthy subjects recruited by Santa Lucia Foundation. Plasma is widely used to assess biomarkers of pathological states in human studies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), a unique technology with the requisite of specificity, sensitivity and quantitative precision capabilities, allowed me to qualify and quantify target sphingolipids.