Analyzing roles of sphingolipid in neurodegenerative blue cheese mutants in drosophila
Date of Issue2014
School of Biological Sciences
We are interested in the involvement of sphingolipids in neurodegeneration in Drosophila. To look at this, we study animals deficient in Blue cheese, a BEACH domain protein involved in autophagy and lysosomal transport, and highly expressed in the nervous system. Blue cheese mutants exhibit shortened life span, extensive neuronal loss and accumulation of ubiquitinated proteins in the brain (Finley et al, 2003). Because of the activity of the BEACH domain in triggering a neutral sphingomyelinase upon inflammatory cytokine signaling and the involvement of sphingolipids in various forms of degeneration in humans we suspected mutation in blue cheese gene could affect sphingolipid balance. In order to test this, we first introduced mutations in the sphingolipid-biosynthetic pathway into the blue cheese background to look for genetic interactions. SMase (sphingomyelinase) and slab (ceramidase) mutants and overexpressors in the background of the blue cheese mutation indeed modulated neuronal survival, in a way that suggests that ceramide levels in the brain are critical. Mass spectrometric quantification of sphingolipid levels in larval brain showed dysregulation of ceramides dependent on diet, particularly sphingadienes and hydroxylated ceramides, which are known regulators of autophagy. Our results indicate that blue cheese mutant have misregulated autophagic flux and increased Atg8 accumulation. Genetic interactors of blue cheese that rescued the degenerative phenotype also fixed the autophagic flux defect. Thus, indicating that ~ 10 ~ regulation of ceramide pools could be sensitive to autophagic flux status. Perturbations in lipid levels of blue cheese mutant also resulted changes in levels and phosphorylation of putative ceramide-regulated targets P-Akt, P-JNK and P-MKK4. Based on the sphingolipid changes and genetic interactions, we propose a possible mechanism whereby blue cheese regulates the activity of sphingolipid synthesis, and acts synergistically with ceramidase, depending on nutrient conditions. Dysregulation of sphingolipid metabolites in mutants appears to additionally alter components of MAPK signaling pathways, which may affect neuronal survival.