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.

The macroautophagy (autophagy) pathway is an ancient and highly conserved intracellular process that occurs in most eukaryote cells. We now understand that autophagy is a complex cellular stress response pathway that serves to sequester and transport intracellular components to the lysosome for degradation and recycling. The unique double-membrane feature of the autophagosome vesicles was characterized over 50 years ago from detailed transmission electron microscopy ( TEM ) imaging studies 1. ; 2. ; 3. . Mutational studies done with yeast starting in the 1990s first characterized the molecular components required to initiate the production and completion of new autophagic vesicles ( AV ) or autophagosomes 4. ; 5. ; 6. ; 7. . Subsequently, molecular genetic studies done in worms, fruit flies and mice have characterized the high level of conservation that occurs among different pathway components. Identification of new factors only found in multicellular organisms underscores the intricate role that autophagy plays in complex physiological processes. The pathway requires the input of two parallel conjugation systems that are centered around two ubiquitin-like proteins called Atg8/MAP-LC3 and Atg12 8. ; 9. . In addition, there is the Atg9:Atg18:Atg2 trafficking system that appears to selectively recruit lipid components to the growing phagophore membrane. Pathway activation and regulation of autophagosome formation requires input from both the Atg1/ULK1 10. ; 11. protein kinase initiation complex as well as a phosphatidylinositol 3-kinase (PI3K) complex 12. ; 13. ; 14. . As additional molecular components of the pathway are identified there is a growing understanding that selective forms of autophagy play significant roles in the maintenance and function of different tissue types, including the nervous system (aggrephagy) and the heart (mitophagy). Insight into the function of autophagy in human disease processes is undergoing a rapid period of growth as our understanding of the role that metabolic disorders (i.e. type-II diabetes and metabolic syndrome) have on long-term autophagic function.

BackgroundS1P3 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 FindingsSpecific 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/SignificanceWe 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.

The therapeutic use of monoclonal antibodies (mAbs) is the fastest growing area of pharmaceutical development and has enjoyed significant clinical success since approval of the first mAb drug in1984. However, despite significant effort, there are still no approved therapeutic mAbs directed against the largest and most attractive family of drug targets: G protein-coupled receptors (GPCRs). GPCRs regulate essentially all cellular processes, including those that are fundamental to cancer pathology, such as proliferation, survival/drug resistance, migration, differentiation, tissue invasion, and angiogenesis. Many different GPCR isoforms are enhanced or dysregulated in multiple tumor types, and several GPCRs have known oncogenic activity. With approximately 350 distinct GPCRs in the genome, these receptors provide a rich landscape for the design of effective, targeted therapies for cancer, a uniquely heterogeneous disease family. While the generation of selective, efficacious mAbs has been problematic for these structurally complex integral membrane proteins, progress in the development of immunotherapeutics has been made by several independent groups. This chapter provides an overview of the roles of GPCRs in cancer and describes the current state of the art of GPCR-targeted mAb drugs.

The Foxp subfamily of forkhead/HNF3 transcription factors has recently been recognized because of its involvement in autoimmune disease, speech and language disorders, and lung development. Domains unique to this subfamily include a divergent DNA-binding winged helix, a leucine zipper, a zinc finger, and a polyglutamine tract. Little is known about the properties of these proteins that are fundamental to their function as transcription factors nor how the Foxp sequence motifs regulate their transcriptional regulatory properties. We report here a structure/function analysis of the Foxp1 protein. We have analyzed the alternative splice isoforms 1A and 1C and also report the cloning and characterization of a novel isoform Foxp1D that lacks the polyglutamine domain. We have isolated the preferred DNA-binding sites for Foxp1 transcription factors. Foxp1A, C, and D isoforms and the related Foxp2 protein repress gene transcription via binding to this consensus site or to a naturally occurring site within the SV40 and the interleukin-2 promoters. In some cases the strength of Foxp1 repression is mediated by the polyglutamine domain. Unlike previously characterized forkhead factors, Foxp1 proteins can form homodimers or heterodimers with subfamily members. The dimerization domain was localized to an evolutionarily conserved C2H2 zinc finger and leucine zipper motif. Finally, we demonstrate that Foxp1, although broadly expressed, is further regulated by tissue-specific alternative splicing of these functionally important sequence domains. These results suggest that Foxp1 proteins have diverse functional roles in different cell and tissue types.