Blocking apoptotic signaling rescues axon guidance in Netrin mutants.

Netrins are guidance cues that form gradients to guide growing axons. We uncover a mechanism for axon guidance by demonstrating that axons can accurately navigate in the absence of a Netrin gradient if apoptotic signaling is blocked. Deletion of the two Drosophila NetA and NetB genes leads to guidance defects and increased apoptosis, and expression of either gene at the midline is sufficient to rescue the connectivity defects and cell death. Surprisingly, pan-neuronal expression of NetB rescues equally well, even though no Netrin gradient has been established. Furthermore, NetB expression blocks apoptosis, suggesting that NetB acts as a neurotrophic factor. In contrast, neuronal expression of NetA increases axon defects. Simply blocking apoptosis in NetAB mutants is sufficient to rescue connectivity, and inhibition of caspase activity in subsets of neurons rescues guidance independently of survival. In contrast to the traditional role of Netrin as simply a guidance cue, our results demonstrate that guidance and survival activities may be functionally related.

Drosophila Vap-33 is required for axonal localization of Dscam isoforms.

Mutations in VAPB have been identified in a familial form of amyotrophic lateral sclerosis (ALS), and reduced VAPB levels have been found in patients with sporadic ALS. Vap protein family members from different species and cell types have been implicated in a number of cellular functions, but how Vap dysfunction in neurons and/or muscles contributes to motor neuron degeneration and death is poorly understood. Using Drosophila as a model organism, we show that Vap physically interacts with and affects the axonal functions of the Down syndrome cell adhesion molecule (Dscam). Dscam is a cell-surface receptor involved in axon and dendritic patterning and neuron self-recognition and avoidance. Alternative splicing of the Dscam transcript leads to the production of Dscam isoforms that contain one of two possible transmembrane (TM) domain and flanking sequences that either restrict the isoform to dendrites and cell bodies (TM1) or target the isoform to axon processes (TM2). We find that Vap specifically interacts with Dscam isoforms that contain the TM2 cytoplasmic juxtamembrane flanking sequences. Using loss-of-function genetics, we further show that Vap is required for localization of Dscam isoforms containing TM2 to axons and that Vap loss suppresses Dscam gain-of-function axon phenotypes. We propose that Vap function is required in neurons to selectively traffic proteins to axons, and disruption of this function may contribute to the pathology of ALS.

Drosophila activated Cdc42 kinase has an anti-apoptotic function.

Activated Cdc42 kinases (Acks) are evolutionarily conserved non-receptor tyrosine kinases. Activating somatic mutations and increased ACK1 protein levels have been found in many types of human cancers and correlate with a poor prognosis. ACK1 is activated by epidermal growth factor (EGF) receptor signaling and functions to regulate EGF receptor turnover. ACK1 has additionally been found to propagate downstream signals through the phosphorylation of cancer relevant substrates. Using Drosophila as a model organism, we have determined that Drosophila Ack possesses potent anti-apoptotic activity that is dependent on Ack kinase activity and is further activated by EGF receptor/Ras signaling. Ack anti-apoptotic signaling does not function through enhancement of EGF stimulated MAP kinase signaling, suggesting that it must function through phosphorylation of some unknown effector. We isolated several putative Drosophila Ack interacting proteins, many being orthologs of previously identified human ACK1 interacting proteins. Two of these interacting proteins, Drk and yorkie, were found to influence Ack signaling. Drk is the Drosophila homolog of GRB2, which is required to couple ACK1 binding to receptor tyrosine kinases. Drk knockdown blocks Ack survival activity, suggesting that Ack localization is important for its pro-survival activity. Yorkie is a transcriptional co-activator that is downstream of the Salvador-Hippo-Warts pathway and promotes transcription of proliferative and anti-apoptotic genes. We find that yorkie and Ack synergistically interact to produce tissue overgrowth and that yorkie loss of function interferes with Ack anti-apoptotic signaling. Our results demonstrate how increased Ack signaling could contribute to cancer when coupled to proliferative signals.