Indeed, crystallographic analysis has confirmed that disease muta

Indeed, crystallographic analysis has confirmed that disease mutations alter the shape of the nucleotide binding pocket, PLX-4720 cell line alter nucleotide loading, and

impair the normal reordering of the N-domain structure that permits cycling between alternative conformations ( Tang et al., 2010). As a consequence, the balance of VCP-adaptor interactions is altered, enhancing the interaction of VCP with some adaptors while diminishing interaction with others ( Fernández-Sáiz and Buchberger, 2010). The central question concerning the pathogenesis of VCP-related disease is which functions of VCP are altered by disease-causing mutations. To address this question in an unbiased way, we generated a Drosophila model that captures VCP mutation-dependent degeneration. Some aspects of this Drosophila model are reminiscent of the phenotypes observed in PTEN-induced putative kinase 1 (PINK1) and parkin mutant flies. Indeed, we demonstrate genetic interactions that place VCP downstream of the PINK1/Parkin pathway in vivo. Mechanistic studies JAK2 inhibitors clinical trials in vitro reveal that VCP is recruited

to mitochondria in a manner that requires Parkin-dependent ubiquitination of mitochondrial proteins. Moreover, VCP is essential to the regulated degradation of membrane proteins, including Mitofusins, and clearance of damaged mitochondria. Most importantly, these studies reveal that this function of VCP is impaired by pathogenic mutations. The species Drosophila melanogaster has a single, highly conserved ortholog of human VCP called dVCP. We developed a Drosophila model of VCP-related

disease by introducing disease-homologous mutations into dVCP. Expression directed to the eye of these animals resulted in mutation-dependent eye degeneration despite equal levels of transgene expression ( Ritson et al., 2010). Expression of wild-type dVCP in motor neurons with the driver OK371-GAL4 did not impact fly viability, whereas motor neuron expression of mutant dVCP resulted in substantial pupal lethality ( Figure 1A). The few adult escapers expressing mutant dVCP in motor neurons died shortly after eclosion. In 3rd-instar larval animals, a mutation-dependent locomotor phenotype was evident, as documented medroxyprogesterone in an assay of larval crawling ( Figure 1B). Evaluation of the neuromuscular junction (NMJ) in these animals revealed a striking mutation-dependent morphological phenotype that included reduced numbers of synaptic boutons, an accumulation of ghost boutons, and reduced density of active zones ( Figures 1C–1E and Figure S1, available online). Evaluation of NMJ morphology in rare surviving mutant dVCP adults also revealed morphological defects including an accumulation of synaptic footprints consistent with denervation ( Figure S2). Consistent with our observations in motor neurons, expression of dVCP in muscle with the driver MHC-GAL4 resulted in mutation-dependent muscle degeneration and a dropped wing phenotype ( Figure 1F and data not shown).

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