Loss of drebrin

NMDA-induced excitotoxicity elicits the degradation of drebrin in primary hippocampal and cortical neurons. This process is triggered by calcium influx and mediated by calpains.

Drebrin is an evolutionarily conserved actin-binding protein in dendritic spines.  Overexpression of drebrin A in neurons enlarges dendritic spines and decreases spine motility, whereas down-regulation of drebrin A in neurons decreases the density and width of dendritic spines and inhibits synaptic clustering of NMDARs.

Drebrin forms stable actin filaments and plays a pivotal role in dendritic spine morphogenesis (Hayashi and Shirao 1999; Takahashi et al., 2003; Takahashi et al., 2006).

During the initial stage of synaptic plasticity (either LTP or LTD), Ca²⁺ influx through the NMDA receptors arises and it brings out drebrin exodus from dendritic spines (Sekino et al., 2006).

Furthermore, prolonged NMDA-induced excitotoxicity induces calpain-mediated degradation of drebrin in vitro and in vivo.

Loss of drebrin from dendritic spines can be detected by immunocytochemistry, ELISA or or Western blotting (Counts et al., 2006; Ishizuka et al., 2014).

The twenty-one-day primary cultured neurons were prepared using frozen stock of dissociated hippocampal neurons (Koganezawa et al, 2023; Hanamura et al 2019). In brief, cells were cultured in multi well microplates with defined medium. 

For immunocytochemistry, the cultured neurons were incubated with chemicals for 1 hour. After fixation, cultured neurons were immunostained with anti-drebrin and anti-MAP2 antibodies. The cluster density of drebrin along the dendrites was automatically quantified using high content analysis instruments (Hanamura et al, 2019, Mitsuoka et al, 2019). 

For enzyme-linked immunoassay, after the incubation of chemicals the extracts of neurons were quantified using ELISA kit for drebrin (Higa et al,2024),

For WB, 

Koganezawa, N., Roppongi, R.T.,Sekino, Y., Tsutsui, I., Higa, A.,Shirao, T. Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons. J. Vis. Exp. (191), e64872, doi:10.3791/64872 (2023).

Mitsuoka T, Hanamura K, Koganezawa N, Kikura-Hanajiri R, Sekino Y, Shirao T. “Assessment of NMDA receptor inhibition of phencyclidine analogues using a high-throughput drebrin immunocytochemical assay” J Pharmacol Toxicol Methods 2019 May 10:106583. doi: 10.1016/j.vascn.2019.106583.

Hanamura K, Koganezawa N, Kamiyama K, Tanaka N, Oka T, Yamamura M, Sekino Y, Shirao T. “High-content imaging analysis for detecting the loss of drebrin clusters along dendrites in cultured hippocampal neurons.” J Pharmacol Toxicol Methods. 2018 Sep - Oct;99:106607. doi: 10.1016/j.vascn.2019.106607.

Chimura T, Launey T, Yoshida N (2015) Calpain-Mediated Degradation of Drebrin by Excitotoxicity In vitro and In vivo. PLoS ONE 10(4): e0125119. doi:10.1371/journal.pone.0125119

Ishizuka, Y. et al. (2014), Histone deacetylase mediates the decrease in drebrin cluster density induced by amyloid beta oligomers. Neurochem Int, 76, 114-121.