Sun-Jung Cho

Presence of translation elongation factor-1A (eEF1A) in the excitatory postsynaptic density of rat cerebral cortex

The postsynaptic density (PSD) is a proteinaceous cellular structure that is specialized for postsynaptic signal transduction. Here, we show that eukaryotic translation factor-1A (eEF1A; formerly known as eEF-1alpha) is associated with the excitatory PSD in rat forebrain. Immunoblot analysis showed that eEF1A in the PSD fraction is enriched over homogenate. Salt (1.0M NaCl), but not non-ionic detergents such as Triton X-100 (1.0%) and n-octyl glucoside (1.0%), could dissociate eEF1A from the PSD core. In cultured cortical neurons, eEF1A was colocalized with postsynaptic markers (PSD95 and SynGAPalpha), but not with a presynaptic marker (synaptophysin). These results indicate that eEF1A is present in the PSD of the excitatory synapses.

Protrusion of N-acetylglucosamine Kinase Clusters into the Base of Excitatory Synapses

N-Acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) catalyzes the phosphorylation of GlcNAc to GlcNAc-6-phosphate (GlcNAc-6-P). Despite detailed characterization of the enzyme itself, there have been few studies on the expression of NAGK in mammalian tissues. In the rat hippocampal neuron in culture, NAGK-immunoreactivity (IR) formed clusters in somatodendritic domains. In this study we characterized the NAGK clusters that protrude out the long axis of dendritic shafts. By double-labeling of the neurons with antibodies against NAGK and various synaptic proteins, we show that NAGK is positioned at the base of spines, while there were no NAGK protrusions into inhibitory postsynaptic sites. Immunoblot analysis showed that NAGK was included in synaptosomes but not in PSD fractions. Our results indicate that the NAGK clusters at the dendritic periphery protrude into spines.

A Reliable Protocol for transfection of mature primary hippocampal neurons using a neuron-glia co-culture system

DNA transfection is a powerful tool for studying gene functions. The Ca 2+ -phosphate precipitation remains one of the most popular and cost-effective transfection techniques. Mature neurons are more resistant to transfection than young ones and most other cell types, and easy to die if microenvironment changes. Here, we report a transfection protocol for mature neurons. The critical modifications are inclusion of glial cells in culture and careful control of Ca 2+ -phosphate precipitation under microscope. Cerebral glial cells were grown until ~70-80% confluence in DMEM/10% horse serum, which was thereafter replaced with serum-free Neurobasal/Ara-C, and E19 hippocampal neurons were plated onto the glial layer. Formation of fine DNA/Ca 2+ -phosphate precipitates was induced using Clontech CalPhos™ Mammalian Transfection Kit, and the size (0.5-1 ㎛ in diameter) and density (about 10 particles/100 ㎛²) were carefully controlled by the time of incubation in the medium. This modified protocol can be reliably applied for transfection of mature neurons that are maintained longer than two weeks in vitro, resulting in 10-15 healthy transfected neurons per a well of 24-well plates. The efficacy of the protocol was verified by punctate expression of pEGFP-CaMKIIα, a synaptic protein, and diffuse expression of pDsRed2. Our protocol provides a reliable method for transfection of mature neurons in vitro.

An efficient and reliable electroelution method from SDS-PAGE: Identification of a 31 kDa protein in the postsynaptic density fraction as adenine nucleotide translocator 1

The molecular composition of the postsynaptic density (PSD) is largely hon. In this report, an electroelution protocol was demonstrated to be used for efficient isolation of PSD proteins with diverse molecular sizes. Using this protocol, a 31 kDa protein in the 1% n-octyl glucoside-insoluble PSD fraction (termed as PSD31) was purified from SDS-gels, and internal peptides were determined for amino acid sequences. The amino acid sequences of the PSD31 were highly homologous with the adenine nucleotide translocator 1 (ANTI). The association of ANTl with PSD suggests presence of a mechanism in synapses for releasing adenosine nucleotides into the extracellular space.