RT Journal Article
SR Electronic
T1 Epileptic encephalopathy-causing mutations in <em>DNM1</em> impair synaptic vesicle endocytosis
JF Neurology Genetics
JO Neurol Genet
FD Lippincott Williams &amp; Wilkins
SP e4
DO 10.1212/01.NXG.0000464295.65736.da
VO 1
IS 1
A1 Ryan S. Dhindsa
A1 Shelton S. Bradrick
A1 Xiaodi Yao
A1 Erin L. Heinzen
A1 Slave Petrovski
A1 Brian J. Krueger
A1 Michael R. Johnson
A1 Wayne N. Frankel
A1 Steven Petrou
A1 Rebecca M. Boumil
A1 David B. Goldstein
YR 2015
UL http://ng.neurology.org/content/1/1/e4.abstract
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