Dissecting Roles for Drosophila TNF and TNF Receptors in Regulating Neuronal Morphology

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In mammals, the Tumour Necrosis Factor Receptor (TNFR) superfamily is composed of 29 members with critical roles in numerous cellular events1. One of the key cellular events regulated by TNFRs is the activation of caspases. In some physiological settings, TNFR-activation causes the assembly of an `apoptosome' that invariably drives apoptosis. In others, TNFRs activate a JNK signaling cascade that can either induce caspase-dependent apoptosis or activate caspases for critical sublethal roles2. The signaling pathways that lead to apoptosome assembly and activation are well understood but the TNFR-driven mechanisms that control JNK-dependent caspase activation remain largely unknown. Indeed, the complexity of mammalian TNFR superfamily members and the downstream signalling paths activated by them has slowed progress in this field. To address this, we initiated a new program using Drosophila melanogaster to identify fundamental signalling events required for receptor-induced JNK-dependent caspase activation. Drosophila have only one TNF-like ligand, Eiger2,3 and only one TNFR receptor, Wengen4. Eiger-induced caspase activation in Drosophila depends on JNK activation; it is therefore a simplified model of TNF-TNFR signaling that can be utilised to characterize the signaling pathway between TNF and JNK activation2,3. Eiger overexpression produces significant cell death even in flies lacking Wengen. We therefore conducted a screen to identify additional Eiger receptors and we have found that Sidekick plays a crucial role in Eiger-induced cell death. Sidekick is a type 1 transmembrane protein recently described as an adhesion molecule5. A separate group has identified Grindenwald as another potential component of this receptor complex6 and we have confirmed that Grindenwald plays an important role in Eiger signalling. In aim one, we will evaluate the interplay between Wengen, Sidekick and Grindenwald in the Eiger signaling pathway. Ubiquitination is a critical post-translational modification required for TNFR signaling. We conducted a series of experiments to elucidate if K63 ubiquitination plays a role in Eiger-Wengen signaling and found that the E2 bendless is essential for Eiger-induced cell death. In independent screens, we (1) identified 18 proteins that became heavily ubiquitinated when the Eiger pathway is activated, and (2) identified 11 E3 ligases require for Eiger-induced death. Intriguingly, from this dataset we found that Eiger signaling results in ubiquitination of histone H2A and that Sex Combs Extra (Sce), the E3 ligase responsible of the mono-ubiquitination of H2A7,8, is required for Eiger-induced cell death and for caspase-dependent pruning of Drosophila ddaC sensory neurons. In aim 2, we will test the hypothesis that Eiger-dependent activation of Sex Combs Extra, and the subsequent ubiquitination of histone H2A, play critical role in Eiger-induced caspase activation.