Abstract
Objective: To elucidate the functional consequences of epileptic encephalopathy–causing de novo mutations in DNM1 (A177P, K206N, G359A), which encodes a large mechanochemical GTPase essential for neuronal synaptic vesicle endocytosis.
Methods: HeLa and COS-7 cells transfected with wild-type and mutant DNM1 constructs were used for transferrin assays, high-content imaging, colocalization studies, Western blotting, and electron microscopy (EM). EM was also conducted on the brain sections of mice harboring a middle-domain Dnm1 mutation (Dnm1Ftfl).
Results: We demonstrate that the expression of each mutant protein decreased endocytosis activity in a dominant-negative manner. One of the G-domain mutations, K206N, decreased protein levels. The G359A mutation, which occurs in the middle domain, disrupted higher-order DNM1 oligomerization. EM of mutant DNM1-transfected HeLa cells and of the Dnm1Ftfl mouse brain revealed vesicle defects, indicating that the mutations likely interfere with DNM1's vesicle scission activity.
Conclusion: Together, these data suggest that the dysfunction of vesicle scission during synaptic vesicle endocytosis can lead to serious early-onset epilepsies.
GLOSSARY
- DAPI=
- 4',6-diamidino-2-phenylindole;
- EDC=
- 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide;
- EM=
- electron microscopy;
- GFP=
- green fluorescent protein;
- LGS=
- Lennox-Gastaut syndrome;
- RFP=
- red fluorescent protein;
- RIPA=
- radioimmunoprecipitation assay;
- TBS=
- Tris-buffered saline;
- WT=
- wild type
Footnotes
Funding information and disclosures are provided at the end of the article. Go to Neurology.org/ng for full disclosure forms. The Article Processing Charge was paid by the authors.
Supplemental data at Neurology.org/ng
- Received March 1, 2015.
- Accepted in final form March 20, 2015.
- © 2015 American Academy of Neurology
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