Abstract
Charcot-Marie-Tooth (CMT) is a group of inherited diseases clinically and genetically heterogenous, characterised by length dependent degeneration of axons of the peripheral nervous system. A missense mutation (p.R158H) in the pyruvate dehydrogenase kinase 3 gene (PDK3) has been identified as the genetic cause for an X-linked form of CMT (CMTX6) in two unrelated families. PDK3 is one of four PDK isoenzymes that regulate the activity of the pyruvate dehydrogenase complex (PDC). The balance between kinases (PDKs) and phosphatases (PDPs) determines the extend of oxidative decarboxylation of pyruvate to generate acetyl CoA, critically linking glycolysis and the energy producing Krebs cycle. We had shown the p.R158H mutation causes hyperactivity of PDK3 and CMTX6 fibroblasts show hyperphosphorylation of PDC, leading to reduced PDC activity and ATP production. In this manuscript we have generated induced pluripotent stem cells (iPSCs) by re-programming CMTX6 fibroblasts (iPSCCMTX6). We also have engineered an isogenic control (iPSCisogenic) and demonstrated that genetic correction of the p.R158H mutation reverses the CMTX6 phenotype. Patient-derived motor neurons (MNCMTX6) show increased phosphorylation of the PDC, energy metabolism defects and mitochondrial abnormalities, including reduced velocity of trafficking mitochondria in the affected axons. Treatment of the MNCMTX6 with a PDK inhibitor reverses PDC hyperphosphorylation and the associated functional deficits founds in the patient motor neurons, demonstrating that the MNCMTX6 and MNisogenic motor neurons provide an excellent neuronal system for compound screening approaches to identify drugs for the treatment of CMTX6.
Highlights
Charcot-Marie-Tooth neuropathy (CMT) is a genetically heterogeneous group of inherited diseases characterised by length dependent axonal degeneration of the motor and sensory neurons of the peripheral nervous system
Our follow up studies demonstrated skin fibroblasts cultured from the original Australian CMTX6 family show increased phosphorylation of the E1 subunit at Ser[293] and Ser[300] sites, causing downregulation of dehydrogenase activity and subsequent accumulation of lactate, reduced production of cellular ATP and mitochondria abnormalities
The discovery of pyruvate dehydrogenase kinase 3 gene (PDK3) as the CMTX6 causative gene has added to the growing list of CMT genes related to mitochondrial biology[5], providing further genetic evidence that mitochondrial pathway deficits may be a common theme of pathogenic mechanism in CMT neuropathies
Summary
Charcot-Marie-Tooth neuropathy (CMT) is a genetically heterogeneous group of inherited diseases characterised by length dependent axonal degeneration of the motor and sensory neurons of the peripheral nervous system. Our laboratory identified a missense mutation (c.G473A p.R158H) in the pyruvate dehydrogenase kinase 3 (PDK3) gene causing an X-linked form of CMT, designated as CMTX63. Experiments using CMTX6 patient fibroblasts demonstrated mutant PDK3 hyperactivity leads to increased phosphorylation of the PDC E1 subunit at specific serine residues and attenuation of the pyruvate dehydrogenase activity. Despite the active research for therapies that can stop or ameliorate degeneration of axons, there is yet no cure for CMT This fact can be explained in part by a reliance on animal models, transformed cell lines and heterologous recombinant systems for drug discovery[7]. In this study we have used patient fibroblasts from a recently identified family carrying the p.R158H PDK3 mutation[4] and, following confirmation of the E1 hyperphosphorylation as a CMTX6 disease signature, generated iPSCs from this patient. We have reversed the CMTX6 cellular phenotype both pharmacologically, using a pan PDK inhibitor, and by genetically correcting the p.R158H PDK3 mutation
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