PT  - JOURNAL ARTICLE
AU  - Ryan S. Dhindsa
AU  - Shelton S. Bradrick
AU  - Xiaodi Yao
AU  - Erin L. Heinzen
AU  - Slave Petrovski
AU  - Brian J. Krueger
AU  - Michael R. Johnson
AU  - Wayne N. Frankel
AU  - Steven Petrou
AU  - Rebecca M. Boumil
AU  - David B. Goldstein
TI  - Epileptic encephalopathy-causing mutations in <em>DNM1</em> impair synaptic vesicle endocytosis
AID  - 10.1212/01.NXG.0000464295.65736.da
DP  - 2015 Jun 01
TA  - Neurology Genetics
PG  - e4
VI  - 1
IP  - 1
4099  - http://ng.neurology.org/content/1/1/e4.short
4100  - http://ng.neurology.org/content/1/1/e4.full
SO  - Neurol Genet2015 Jun 01; 1
AB  - 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.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