Mitochondrial dysfunction is a common cause of peripheral neuropathy. myelin lipid components as well as in accumulation of acylcarnitines an intermediate of fatty acid β-oxidation. Importantly SB-705498 we show that acylcarnitines are released from SCs and induce axonal degeneration. A maladaptive integrated stress response as well as altered SC lipid metabolism are thus underlying pathological mechanisms in mitochondria-related peripheral neuropathies. from a tissue of interest results in severe mtDNA depletion and mitochondrial respiratory chain deficiency (Larsson et al. 1998 Silva et al. 2000 Viader et al. 2011 This makes the tissue-specific deletion of Tfam an effective way to induce mitochondrial dysfunction in a selected population of cells. As detailed elsewhere (Viader et al. 2011 we achieved highly selective and efficient excision of from SCs by mating mice with alleles (ISR induction assays SCs were initially seeded onto collagen coated 24-well plates (~75 0 cells/well) in 10% FBS-DMEM media supplemented with SB-705498 2 μM forskolin and 20 μg/ml of bovine pituitary extract. 48 h later cells were switched to 1% FBS-DMEM media for 2 days to stop proliferation. At this point SCs were treated with either vehicle 5 μM CCCP 2.5 μM oligomycin 10 μM antimycin or 1 μM tunicamycin. RNA or protein were isolated 24 h later. Reported results are from duplicate wells from at least three independent assays. eIF2α kinase shRNA knockdown in 3T3 cells SB-705498 and in vitro ISR induction NIH 3T3 cells were infected with lentivirus expressing shRNA to one of the four eIF2α kinases (HRI PKR PERK GCN2; see SI for details). The infected cells were selected by growth in puromycin for 5 days and cell populations with significant knockdown of each of the kinases were obtained and frozen as ‘polyclonal populations’. Polyclonal populations of cells were then seeded onto 24-well plates (~50 0 cells/well) in 10% FBS-DMEM PRKM8IP media. Sixteen hr after seeding cells were treated with either vehicle or 5 μM CCCP for 3 hrs (for p-eIF2α induction) or 6 hrs (for DDIT3/CHOP induction). Cells were then harvested for Western Blot analysis. Acyl-carnitine release measurements To measure the ability of Tfam-deficient SCs to secrete long-chain acylcarnitines nerves were explanted from Tfam-SCKO and Ctrl mice and maintained in 100 μl of 10% fetal bovine serum (FBS) supplemented with 2 mM L-glutamine and 100 ng/ml of nerve growth factor for 2.5 days. At this time media was collected and immediately frozen in liquid nitrogen. Media was then analyzed for acylcarnitine species content (C2-C18 saturated unsaturated and hydroxylated) as butyl esters by direct flow injection and precursor ion scanning on an API 3200 LC-MS/MS system (Applied Biosystems). Quantitation was achieved using a cocktail of internal standards. Concentrations were normalized to tissue weight. DRG neuron culture and Fluo-4 imaging mouse DRG neurons isolated from E12 embryos were seeded onto either 24-well or 96-well cell cultures plates coated with poly-d-lysine (Sigma) and Laminin (Invitrogen) SB-705498 and all experiments were carried out 5-6 days after seeding. For calcium imaging experiments neurons were incubated with the calcium indicator Fluo-4 AM (2 μM Invitrogen) and neurons were then treated either with vehicle palmitoyl-carnitine (Sigma) or palmitate (Sigma) at the appropriate concentrations. Phase and fluorescence images were acquired every 15 minutes for up to 6 hours using an Operetta imaging system equipped with an environmental chamber (Perkin Elmer) and automated image analysis was carried SB-705498 out using image J. To examine the effect of chronic acylcarnitine exposure DRG neurons were treated daily for up to nine days with vehicle or with palmitoyl-carnitine at the appropriate concentration (see SI for details). Statistical analysis All values are expressed as mean ± SEM and if no units are specified are expressed SB-705498 as percent of control. If not stated otherwise values were determined by unpaired two-tailed Student’s test. All statistical analyses were performed using Microsoft Excel 2007. ? Highlights A mouse model to interrogate how SCs contribute to mitochondria-related neuropathies. Mitochondrial dysfunction in SCs activates a maladaptive integrated stress response. Mitochondrial dysfunction disrupts SC lipid metabolism and depletes.