Primary neuronal cultures of cerebral cortex prepared from E18 embryos of Rats were used after 12 to 17 DIV and transfected with plasmid DNA using Magnetofectamine using conditions settled by Buerli et al (Nature Protocol, 2007)
This paper demonstrated the high efficiency of Magnetofectamine from OZ Biosciences to transfect primary hippocampal neurons. Numerous neuron types can efficiently be transfected using Magnetofection technology.article reference: J Biol Chem. 2014 Jan 30.
Proteasomal Degradation of γ-Aminobutyric AcidB Receptors is Mediated by the Interaction of the GABAB2 C Terminus with the Proteasomal ATPase Rtp6 and Regulated by Neuronal Activity.
Abstract
Regulation of cell surface expression of neurotransmitter receptors is crucial for determining synaptic strength and plasticity but the underlying mechanisms are not well understood. We previously showed that proteasomal degradation of GABAB receptors via the endoplasmic reticulum (ER) associated protein degradation (ERAD) machinery determines the number of cell surface GABAB receptors and thereby GABAB receptor-mediated neuronal inhibition. Here we show that proteasomal degradation of GABAB receptors requires the interaction of the GABAB2 C terminus with the proteasomal AAA-ATPase Rpt6. A mutant of Rpt6 lacking ATPase activity prevented degradation of GABAB receptors but not the removal of Lys48-linked ubiquitin from GABAB2. Blocking ERAD activity diminished the interaction of Rtp6 with GABAB receptors resulting in increased total as well as cell surface expression of GABAB receptors. Modulating neuronal activity affected proteasomal activity and correspondingly the interaction level of Rpt6 with GABAB2. This resulted in altered cell surface expression of the receptors. Thus, neuronal activity-dependent proteasomal degradation of GABAB receptors by the ERAD machinery is a potent mechanism regulating the number of GABAB receptors available for signaling and is expected to contribute to homeostatic neuronal plasticity.
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