Abstract
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.
Abbreviations
- 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