Authors

Koh, J. Y. Iwabuchi, S. Harata, N. C.

Affiliations

Department of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine, Iowa City, IA, USA.

Application Area

Neuroscience

General Fluorescence Microscopy

Abstract

Presynaptic functions of the mammalian central neurons are regulated by a network of protein interactions. Synaptic vesicle recycling in and neurotransmitter release from the presynaptic nerve terminals are altered when a glutamate-deleting mutation is present in the torsinA protein (δE-torsinA). This mutation is linked with a hereditary form of the movement disorder dystonia known as DYT1 dystonia. Although torsinA expression is prevalent throughout the central nervous system, its subcellular localization - in particular with respect to presynaptic nerve terminals - remains unclear. This information would be useful in narrowing down possible models for how wild-type torsinA affects presynaptic function, as well as the nature of the presynaptic dysfunction that arises in the context of δE-torsinA mutation. Here we report on an analysis of the presynaptic localization of torsinA in cultured neurons obtained from a knock-in mouse model of DYT1 dystonia. Primary cultures of neurons were established from heterozygous and homozygous δE-torsinA knock-in mice, as well as from their wild-type littermates. Neurons were obtained from the striatum, cerebral cortex and hippocampus of these mice, and were subjected to immunocytochemistry. This analysis revealed the expression of both proteins in the somata and dendrites. However, neither the nerve terminals nor axonal shafts were immunoreactive. These results were confirmed by fluorogram-based quantitation. Our findings indicate that neither the wild-type nor the δE-torsinA mutant protein is present at substantial levels in the presynaptic structures of cultured neurons. Thus, the effects of torsinA, in wild-type and mutant forms, appear to influence presynaptic function indirectly, without residing in presynaptic structures.

Extract

… “Alexa Fluor 405 was excited using a 400-nm light-emitting diode (LED) (CoolLED-Custom Interconnect)”…

Product Associated Features

Precis Excite: A simple-to-use, multi-wavelength Illumination System. Now superseded by the pE-4000 Illumination System.

Diascopic Technique

DIC

Live Cell Issues

-

Product Type

-

Journal

Neuroscience

Year of Publication

2013

Country of Publication

USA