However, further analysis of these fibroblasts did not detect any alterations in mitochondrial function or ER stress signalling (data not shown). the reduction of this signalling is definitely neuroprotective, independently of defective mitochondria. A video abstract for this article is available online in the supplementary info Recently, endoplasmic reticulum (ER) stress, and in particular dysregulation of the protein kinase R-like endoplasmic reticulum kinase (PERK) branch of the unfolded protein response (UPR) have emerged as major toxic processes in protein misfolding neurodegenerative disorders (examined in Halliday and Mallucci1). Overactivation of PERK signalling is definitely a feature of post-mortem brains of individuals with Alzheimer’s and Parkinson’s diseases and the tauopathies, frontotemporal dementia (FTD) and Progressive Supranuclear Palsy (examined in Scheper and Hoozemans2). In mice with prion disease3 and FTD-like pathology,4 sustained activation of the PERK branch of the UPR leads to chronic reduction in global protein synthesis rates in the brain. The reduction in translation of vital proteins leads to neuronal death, which is rescued by inhibition of the pathway at the level of PERK3, 4, 5 or downstream effectors.6 In Parkinson’s disease (PD), mitochondrial dysfunction, due to loss of function of PTEN-induced putative kinase 1 (PINK1) or PARKIN, is a central pathogenic process (examined in Celardo or XCT 790 mutants show neurodegeneration, a crushed thorax phenotype and mitochondrial dysfunction.9, 10 We therefore asked: first, whether ER pressure occurs in models of PD and contributes to the neurodegenerative phenotype, and second: to what extent, if any, ER pressure is driven by defective mitochondria? We found that mitochondrial dysfunction in or mutant flies XCT 790 does activate the PERK branch of the UPR through the formation of mitofusin bridges between defective mitochondria and the ER. Further, we found that inhibiting PERK signalling genetically and pharmacologically, or through the reduction of mitofusin bridges was neuroprotective in and mutant flies, irrespective of the persistence of defective mitochondria. Results and mutants display activation of the PERK branch of the UPR We 1st examined and mutants for evidence of ER stress and UPR activation. We found increased levels of chaperone-binding immunoglobulin protein (BiP), a marker for ER stress activation, in the body wall muscle mass cells11 of both and mutant larvae compared with wild-type settings (Number 1a). Upon ER stress, BiP dissociates from PERK, which dimerizes and autophosphorylates. XCT 790 Phospho-PERK in turn phosphorylates eukaryotic initiation element 2 alpha (eIF2in and mutants, which were reduced upon knockdown of (Number 1b), consistent with its activation through PERK signalling and raised levels of BiP. Open in a separate window Number 1 Activation of phospho-eIF2signalling and attenuation of translation in and mutant flies. XCT 790 (a) Improved levels of BiP in the body wall muscle mass of and mutant larvae. Representative confocal images with the indicated genotype stained with in and mutant flies are reduced by knockdown of are demonstrated at the top. (c) Polysomal distribution of mRNAs of young adult male flies showing individual ribosomal subunits and the polysome peaks. RNA concentrations were measured from the low (Lo) and high (Hi) translation fractions (meanS.D., asterisks, one-way ANOVA with Dunnett’s multiple assessment test; and mutant flies. XCT 790 Whole-fly lysates were analysed with an anti-puromycin antibody and equal protein loading was assessed by Ponceau S staining of the membranes. Genotypes for (b) Control: was driven by and mutants by KLHL11 antibody polysomal profiling (Number 1c), consistent with a decrease in global translation rates. Additionally, we recognized a decrease in protein synthesis, measured by assessing the incorporation of puromycin, a Tyr-tRNA mimetic, into newly translated proteins (Number 1d).12 These findings support activation of signalling through the PERK branch of ER stress in and mutant flies. and mutants display an enhanced association between defective mitochondria and the ER We next asked whether there is cross-talk between dysfunctional mitochondria and activation of PERK signalling. Red1 and Parkin mediate the ubiquitination and degradation of the profusion element mitofusin (dMfn) within the outer surface of mitochondria; and or mutant flies display an accumulation of dMfn.13 Mitofusin modulates mitochondrial fusion and the tethering of these organelles to the ER.14 To test whether the accumulation of dMfn in both and mutants affected the proximity between mitochondria and the ER, we quantified mitochondriaCER contacts using a previously explained assay. 15 We 1st confirmed the previously reported build up of dMfn in and mutant flies, which could become partially reversed upon RNA interference (RNAi) (Number 2a). Ultrastructural analysis of take flight brains exposed that both and mutants display.