GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 in a redoxdependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. Changes in the actin cytoskeleton also disrupt mitochondria-ER contact sites. This results in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, these findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology. ### Competing Interest Statement The authors have declared no competing interest. * CMT : Charcot-Marie-Tooth DRP1 : Dynamin-related protein 1 GDAP1 : ganglioside-induced differentiation associated protein 1 AR : axonal-recessive ER : endoplasmic reticulum MERCS : mitochondria-ER contact sites GST : glutathione-transferase iPSCs : induced pluripotent stem cells NPCs : neural precursor cells Δψm : mitochondrial membrane potential TMRM : tetramethylrhodamine methyl ester KD : knockdown ETS : electron transfer system netR : phosphorylating respiration TEM : transmission electron microscopy bFGF : basic fibroblast growth factor FCS : fetal calf serum RT : room temperature ICC : Immunocytochemistry PBS-T : PBS-Tween 20 Ex : Excitation Em : Emission PCP : phosphorylation-control-protocol R : routine respiration Omy : oligomycin L : leak respiration FCCP : carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone E : ETS capacity Rot : rotenone Ama : antimycin A ROX : non-mitochondrial residual oxygen consumption BTeam : BRET-based ATP biosensor NLuc : NanoLuciferase 2DG : 2-Deoxy-D-Glucose YFP : Yellow Fluorescent Protein CRISPR-Cas9 : Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR-associated protein 9 gRNA : guided-RNA NeoR : Neomycin resistance gene SDS : sodium dodecyl sulfate SDS-PAGE : sodium dodecyl sulfate polyacrylamide gel electrophoresis DTT : Dithiothreitol SD : standard deviation SEM : standard error of the mean Ctrl : control OXPHOS : oxidative phosphorylation LDHA : lactate dehydrogenase A GDH : glutamate dehydrogenase TCA : tricarboxylic acid cycle PDH : Pyruvate dehydrogenase PDC : Pyruvate dehydrogenase complex PDK3 : pyruvate dehydrogenase kinase isoenzyme 3 MCU : Mitochondrial Calcium uniporter FRET : Förster resonance emission transfer IP3R : inositol trisphosphate receptors