Anti-FADD polyclonal antibody [Alexa Fluor® 555]

Background Despite the plethora of sequence variants in LRRK2, only a few clearly segregate with PD. Even within this group of pathogenic mutations, the phenotypic profile can differ widely. Objective We examined multiple properties of LRRK2 behavior in cellular models over-expressing three sequence variants described in Greek PD patients in comparison to several known pathogenic and non-pathogenic LRRK2 mutations, to determine if specific phenotypes associated with pathogenic LRRK2 can be observed in other less-common sequence variants for which pathogenicity is unclear based on clinical and/or genetic data alone. Methods The oligomerization, activity, phosphorylation, and interaction with FADD was assessed in HEK293T cells over-expressing LRRK2; while the induction of neuronal death was determined by quantifying apoptotic nuclei in primary neurons transiently expressing LRRK2. Results One LRRK2 variant, A211V, exhibited a modest increase in kinase activity, whereas only the pathogenic mutants G2019S and I2020T displayed significantly altered auto-phosphorylation. We observed an induction of detergent-insoluble high molecular weight structures upon expression of pathogenic LRRK2 mutants, but not the other LRRK2 variants. In contrast, each of the variants tested induced apoptotic death of cultured neurons similar to pathogenic LRRK2 in a FADD-dependent manner. Conclusions Overall, despite differences in some properties of LRRK2 function such as kinase activity and its oligomerization, each of the LRRK2 variants examined induced neuronal death to a similar extent. Furthermore, our findings further strengthen the notion of a convergence on the extrinsic cell death pathway common to mutations in LRRK2 that are capable of inducing neuronal death.

Fas (CD95) signalling is best known for its role in apoptosis, however, recent reports have shown it to be involved in other cellular responses as well, including inflammation. Fas and its adaptor protein FADD are known to negatively regulate LPS-induced proinflammatory responses, but their role in LPS-induced type I interferon production is unknown. Here, we demonstrate that Fas engagement on macrophages, using an agonistic Fas antibody CH11, augments LPS-induced NF-kappa B responses, causing increased production of TNF alpha, IL-8, IL-6 and IL-12. Conversely, costimulation with both LPS and CH11 causes a significant reduction in the level of interferon-beta (IFN beta) production. This differential effect involves the Fas adaptor FADD because while LPS-induced IL-6 production increased in FADD(-/-) murine embryonic fibroblasts, LPS-induced IFN beta production was significantly reduced in these cells. Overexpression of a dominant negative form of FADD (FADD-DD) inhibits LPS-induced IFN beta luciferase but not LPS-induced NF-kappa B luciferase. In contrast, overexpression of full-length FADD inhibited LPS-induced NF-kappa B luciferase activation but was seen to augment LPS-induced IFN beta luciferase. Moreover, FADD-DD inhibits TRIF-, TRAM-, IKK epsilon-, TBK-1- and TRAF3-induced IFN beta luciferase production, with coimmunoprecipitation experiments demonstrating an interaction between FADD and TRIF. These data identify FADD as a novel component of the noncanonical Toll-like receptor 4/IFN beta signalling pathway and demonstrate that both Fas and its adaptor FADD can differentially regulate the production of LPS-induced proinflammatory cytokines and type I interferons.