Energy metabolism and mitochondrial defects in X-linked Charcot-Marie-Tooth (CMTX6) iPSC-derived motor neurons with the p.R158H PDK3 mutation

Abstract

PDK3 negatively regulates the pyruvate dehydrogenase complex (PDC) activity in the mitochondria by reversible phosphorylation of its first catalytic component (E1). PDK3 hence has a fundamental role in linking glycolysis to the energy producing Krebs cycle. The discovery of PDK3 has added to the growing list of CMT genes related to mitochondrial biology 5 , suggesting mitochondrial pathway deficits may be a common theme in some CMT neuropathies. Our previous investigations showed the p.R158H mutation produces a PDK3 enzyme with increased kinase activity 3. Experiments using CMTX6 patient fibroblasts demonstrated mutant PDK3 hyperactivity leads to increased phosphorylation of the PDC E1 subunit at specific serine residues and hence attenuation of the pyruvate dehydrogenase activity. Consequently, CMTX6 patient fibroblasts show increased lactate, decreased ATP and alteration of the mitochondrial network. Importantly, E1 hyperphosphorylation was reversed by treating the patient fibroblasts with a pan PDK inhibitor, dichloroacetic acid (DCA), opening a venue for therapeutic intervention for CMTX6 6. 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. The use of human induced pluripotent stem cell (iPSC) technology has recently opened up the possibility to produce disease-relevant human models for drug discovery for inherited diseases in general and neurodegenerative disorders in particular 8. iPSC lines from CMT patients have increasingly been generated 9-11 and key pathological features for the disease have been replicated in some instances 12-15 in CMT patient derived motor neurons. 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. To eliminate the influence of variable genetic backgrounds from genetically unrelated controls, we also generated an isogenic wild type iPSC line by targeted gene correction using the CRISPR/Cas9 system. Our results show the E1 hyperphosphorylation is maintained in the CMTX6-derived iPSCs following reprograming of the patient fibroblasts and is also observed after differentiation into spinal cord motor neurons. Our data reveals abnormalities in the bioenergetic profile and mitochondrial morphological features in the CMTX6-derived motor neurons. Additionally, analyses of the organelle trafficking demonstrated the PDK3 mutation specifically affects mitochondrial trafficking in the patient motor neurons. Importantly, we have reversed the CMTX6 cellular phenotype both pharmacologically, using a pan PDK inhibitor, and by genetically correcting the p.R158H PDK3 mutation. Material and Methods Research guidelines and regulations. All research and cell culture procedures were conducted follow...

Induced pluripotent stem cell-derived motor neurons of CMT type 2 patients reveal progressive mitochondrial dysfunction

“…Of note, an altered mitochondrial metabolic defect was recently reported in iPSC-derived motor neurons from CMTX6 patients. 26 These results indicate that mitochondrial energy deficits could form a common hallmark of axonal CMT neuropathies. Given the length of these neurons, which can be up to 1 m in adults, this may be further exacerbated by axonal transport defects.…”

“…Previous studies have reported axonal transport defects such as decreased mitochondrial speed in iPSC-derived neurons of CMT2A, CMT2E or CMTX6. 20 , 26 However, it remains unknown whether these hallmarks of axonal degeneration are common to all CMT2 subtypes. To verify whether this was at least a shared feature in our five CMT2 lines, we performed live-cell imaging experiments to study mitochondrial and lysosomal trafficking in both control and patient iPSC-derived motor neurons over time using MitoTracker TM and LysoTracker TM dyes, respectively.…”

Dysregulation of PGC-1α-Dependent Transcriptional Programs in Neurological and Developmental Disorders: Therapeutic Challenges and Opportunities

“…While recent studies suggest that deriving neurons directly from fibroblasts without going through the process of iPSC reprogramming can avoid rejuvenation effects of the culture process [ 320 , 323 ], it is still important to consider that these cell culture systems may not fully reproduce the metabolic or mitochondrial state of differentiated cells in vivo [ 324 , 325 ], as neurons derived from iPSCs exhibit a higher glycolytic activity than mouse primary cultures [ 326 ]. However, some studies indicate that metabolic alterations due to disease state can still be maintained in neurons derived from patient iPSCs [ 268 , 327 ]. Even so, caution should be taken when using these types of cells to identify novel compounds which engage processes normally associated with differentiated cell types.…”

Modelling Mitochondrial Disease in Human Pluripotent Stem Cells: What Have We Learned?

“… [ 104 , 119 , 120 , 123 , 211 ] OL-iPSC c.[(2496+1G>T)];[=] − PD-OPA1 G488R (2 clones) p.[G488R];[=] + − PD-OPA1 A495V #72 p.[A495V[;[=] + − Opa1P (2 clones) c.[(33-34ins9)];[=] − − Opa1 (2 clones) c.[(33-34ins9)];[=] − − OPA1+/− hESC N/D (haploinsufficiency) + CMTX6 (PDK3) iPSCCMTX6 p.[(R158H)];[0] + SpMN ↓ ATP c,d Fragmented mitochondrial network Mitochondrial trafficking defect PDC E1 hyperphosphorylation Treatment with the pan PDK inhibitor DCA reduced PDC E1 hyperphosphorylation, and improved mitochondrial fragmentation and motility. [ 216 ] Leigh-like syndrome (PMPCB) DII-2 iPSC (3 clones) p.[I422T];[I422T] − − − NESC N/D N/D Inefficient processing and accumulation of intermediate Fra...…”