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Pathogenic Mechanisms Underlying X-Linked Charcot-Marie-Tooth Neuropathy (CMTX6) in Patients with a Pyruvate Dehydrogenase Kinase 3Mutation

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Pathogenic Mechanisms Underlying X-Linked Charcot-Marie-Tooth Neuropathy (CMTX6) in Patients with a Pyruvate Dehydrogenase Kinase 3Mutation Neurobiology of Disease 94 (2016) 237–244 Contents lists available at ScienceDirect Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi Pathogenic mechanisms underlying X-linked Charcot-Marie-Tooth neuropathy (CMTX6) in patients with a pyruvate dehydrogenase kinase 3mutation Gonzalo Perez-Siles a,c,⁎,CarolynLya, Adrienne Grant a, Alexander P. Drew a, Eppie M. Yiu d,e,f, Monique M. Ryan d,e,f, David T. Chuang g, Shih-Chia Tso g, Garth A. Nicholson a,b,c,MarinaL.Kennersona,b,c,⁎ a Northcott Neuroscience Laboratory, ANZAC Research Institute, University of Sydney, Concord, NSW, Australia b Molecular Medicine Laboratory, Concord Hospital, Concord, NSW, Australia c Sydney Medical School, University of Sydney, Sydney, NSW, Australia d Department of Neurology, Royal Children's Hospital, Flemington Road, Parkville, VIC, Australia e Neuroscience Research, Murdoch Childrens Research Institute, Melbourne, VIC, Australia f Department of Pediatrics, The University of Melbourne, VIC, Australia g Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA article info abstract Article history: Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. An X-linked form of Received 11 March 2016 CMT (CMTX6) is caused by a missense mutation (R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 Revised 22 June 2016 (PDK3) gene. PDK3 is one of 4 isoenzymes that negatively regulate the activity of the pyruvate dehydrogenase Accepted 3 July 2016 complex (PDC) by reversible phosphorylation of its first catalytic component pyruvate dehydrogenase (designat- Available online 5 July 2016 ed as E1). Mitochondrial PDC catalyses the oxidative decarboxylation of pyruvate to acetyl CoA and links glycol- ysis to the energy-producing Krebs cycle. We have previously shown the R158H mutation confers PDK3 enzyme Keywords: fi R158H X-linked Charcot-Marie-Tooth neuropathy hyperactivity. In this study we demonstrate that the increased PDK3 activity in patient broblasts (PDK3 ) Pyruvate dehydrogenase kinase 3 leads to the attenuation of PDC through hyper-phosphorylation of E1 at selected serine residues. This hyper- Pyruvate dehydrogenase complex phosphorylation can be reversed by treating the PDK3R158H fibroblasts with the PDK inhibitor dichloroacetate Mitochondria (DCA). In the patient cells, down-regulation of PDC leads to increased lactate, decreased ATP and alteration of Patient fibroblasts the mitochondrial network. Our findings highlight the potential to develop specific drug targeting of the mutant Dichloroacetic acid PDK3 as a therapeutic approach to treating CMTX6. © 2016 Published by Elsevier Inc. 1. Introduction disease and the cellular processes of the peripheral nerve degeneration. However, there are still no effective treatment therapies for CMT. Hereditary motor and sensory disorders of the peripheral nerve The identification of mutations in the pyruvate dehydrogenase ki- form one of the most common groups of human genetic diseases, collec- nase isoenzyme 3 (PDK3) gene as a cause of an X-linked dominant tively called Charcot–Marie–Tooth (CMT) neuropathy. CMT is a clinical- form of CMT (CMTX6) (Kennerson et al., 2013)(Kennerson et al., ly and genetically heterogeneous disorder affecting 1 in 2500 people 2016) has added to the growing list of CMT genes related to the biology (Fowler et al., 1997). Clinical features include progressive weakness of mitochondria (Pareyson et al., 2015) suggesting that pathways lead- and atrophy of distal muscles, high arched feet (pes cavus) and loss of ing to mitochondrial deficits may be a common theme in inherited axo- deep tendon reflexes. Mutations in N80 genes cause CMT and related nal neuropathies. The nuclear-encoded pyruvate dehydrogenase disorders. The diversity of the cellular and molecular function of pro- complex (PDC) is located in the mitochondrial matrix and catalyses teins implicated in CMT is providing insight to the pathophysiology of the conversion of pyruvate to acetyl CoA, a key regulatory step of the en- ergy-producing Krebs cycle. The mammalian PDC is a 9.5 million-Dalton protein machine comprising multiple copies of pyruvate dehydroge- nase (E1), dihydrolipoyl transacetylase (E2), dihydrolipoamide dehy- ⁎ Corresponding authors at: Northcott Neuroscience Laboratory, ANZAC Research drogenase (E3), and the E3-binding protein (E3BP) (Reed, 2001). PDC Institute, University of Sydney, Concord, NSW, Australia. is regulated through reversible phosphorylation of the E1 subunit of E-mail addresses: [email protected] (G. Perez-Siles), [email protected] (M.L. Kennerson). PDC by the four PDK isoenzymes (PDK1 to PDK4) that act to inactivate Available online on ScienceDirect (www.sciencedirect.com). the PDC (Korotchkina and Patel, 1995) and dephosphorylation by http://dx.doi.org/10.1016/j.nbd.2016.07.001 0969-9961/© 2016 Published by Elsevier Inc. 238 G. Perez-Siles et al. / Neurobiology of Disease 94 (2016) 237–244 pyruvate dehydrogenase phosphatases (PDPs) restores PDC activity detection at 450 nm. Briefly, 1 × 106 fibroblasts were trypsinized and re- (Huang et al., 1998). suspended in PBS containing proteinase inhibitors (cOmplete, Mini Pro- Previously we have shown that the R158H mutation confers PDK3 tease Inhibitor, Roche). After protein quantification, the concentration hyperactivity and binds with stronger affinity than its wild-type coun- was adjusted to 10 mg/ml using PBS. Intact functional PDC was solubi- terpart to the inner-lipoyl (L2) domain of the E2 component of PDC lized by adding a detergent (1:9 volumes) provided in the kit. Using (Kennerson et al., 2013). In this study, we further explore the down- the 1XBuffer, sample concentration was adjusted to 1 mg/ml and pro- stream consequences of the R158H mutation, using primary fibroblasts tein (200 μg per well) was assayed in triplicate and incubated at RT (PDK3R158H) cultured from a CMTX6 patient. We demonstrate the im- for 3 h. Wells were washed twice with 1× Stabilizer reagent and the pact of the PDK3 mutation on the phosphorylation status of the E1 sub- Assay solution (200 μl) was added to the wells. Using an EnSpire Multi- unit, its consequences on the PDC activity as well as the subsequent mode Plate Reader (PerkinElmer) the assay absorbance at 450 nm was biochemical outcomes and the impact on mitochondrial morphology measured at RT for 30 min at 30 s intervals. in the PDK3R158H cells. 2.5. Lactate production assay 2. Methods Prior to the experiment (72 h), 1 × 105 cells per well were seeded in 2.1. Fibroblasts culture a P6 well culture plate. Lactate production was evaluated using the Lac- tate Colorimetric Assay Kit II (BioVision). On the day of the experiment, Patients participating in this study provided written consent accord- media containing 10% (v/v) FBS was replaced with DMEM and after a ing to protocols approved by the Sydney Local Health District Human 4 h incubation media (1 ml) was collected and kept on ice. A dilution Ethics Review Committee, Concord Repatriation General Hospital, Syd- (1 in 10) in Assay Buffer (50 μl) plus 50 μl of Reaction Mix was added fi ney, Australia (reference number: HREC/11/CRGH/105). Primary bro- to the ELISA plate. The reaction was incubated for 30 min at RT. The ab- blasts were cultured from patient skin biopsies and maintained with sorbance at 450 nm was measured with an EnSpire Multimode Plate fi broblast cell culture medium: DMEM (Gibco, Life technologies) sup- Reader (PerkinElmer). Levels of lactate were corrected per mg of pro- plemented with 10% (v/v) fetal bovine serum (SAFC Biosciences), 1% tein. Protein concentration was determined using the Pierce BCA Pro- (v/v) Penicillin Streptomycin (Gibco, Life technologies) and 1% (v/v) tein Assay Kit (ThermoScientific). L-glutamine (Gibco, Life technologies) and maintained at 37 °C in hu- midified air and 5% CO . 2 2.6. ATP assay 2.2. Real-time (RT) quantitative PCR Prior to the experiment (24 h), 2 × 104 cells per well were plated in P96 well plates. Cellular ATP was measured using an ATPlite assay kit RNA was extracted from fibroblasts using the RNeasy mini kit (PerkinElmer, Massachusetts, UK). Briefly, mammalian cell lysis solu- (Qiagen). Reverse transcribed template was prepared using the High- tion (50 μl) was added to 100 μl cell suspension per well and the plate Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quanti- incubated for 10 min. Substrate solution (50 μl) was added to the cell ly- tative RT-PCR was performed using the PDK3-specificTaqManGeneEx- sate and incubated in the absence of light for 10 min. Luminescence was pression Assay Hs00178440_m1 (Applied Biosystems) and run on a measured on an EnSpire Multimode Plate Reader (PerkinElmer). Data is StepOnePlus™ real-time PCR System (Applied Biosystems). The com- represented as the nmoles of ATP detected for the 2 × 104 cells assayed. parative 2−ΔΔCt method (Schmittgen and Livak, 2008) was used to mea- The average of the signal was determined for the 8 replicates plated for sure the relative quantitation of PDK3 expression in affected and control each cell line. individuals. 18S (Hs99999901_s1; Applied Biosystems) was used as the housekeeping gene. 2.7. Assessment of mitochondrial morphology 2.3. Immunofluorescence Prior to the experiment (48 h) 3 × 104 cells per well were plated on Control and patient fibroblasts were incubated with 200 nM cover slips and placed in P24 well plates. The mitochondrial network in fi MitoTracker Red CMX Red (Invitrogen) to visualize mitochondrial control and patient broblasts was stained with 200 nM MitoTracker fi structures. Cells were fixed with 4% formaldehyde, permeabilized in Red CMX Red for 30 min. Cells were xed and mounted as previously fi phosphate-buffered saline (PBS) containing 0.3% (v/v) Triton X-100 described. N = 30 individual well de ned cells were analyzed per sam- and blocked in 3% (w/v) bovine serum albumin (BSA). Cells were incu- ple. Using the image processing package Fiji (Schindelin et al., 2012), fi bated with affinity-purified anti-pSer293 (phosphorylation site 1), anti- images were binarised by conversion to 8 bit image types.
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