Premium
Novel Bryostatin‐1 Targets: Mammalian Unc13‐1 and Unc13‐2 Isoforms
Author(s) -
Blanco Francisco A,
Pany Satyabrata,
Ghosh Anamitra Alejandro,
You Younki,
Das Joydip
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.760.11
Subject(s) - protein kinase c , microbiology and biotechnology , green fluorescent protein , bryostatin 1 , transfection , hippocampal formation , gene isoform , diacylglycerol kinase , biology , chemistry , biochemistry , signal transduction , neuroscience , gene
Neuroprotective Bryostatin‐1 (Bryo) is currently under phase II clinical trials for Alzheimer's disease (AD). Bryo has been postulated to enact its neuroprotective properties through the modulation of protein kinase C (PKC) by interacting with the diacylglycerol/phorbol ester‐binding pocket of its C1 domain. Mammalian un13‐1 (Munc13‐1) and brain specific unc13‐2 (bMunc13‐2) contain C1 domain with a high degree of homology to that of PKC. Additionally, Munc13‐1 and bMunc13‐2 are expressed in the hippocampus and function in synaptic vesicular priming. In this study we explored the existence of an interaction between Bryo and these Munc13 isoforms. The long‐term goals of this project are to expand the understanding of Munc13‐C1‐domain and identify novel effectors of Bryo. We hypothesized that Bryo would interact with Munc13‐1 and bMunc13‐2, resulting in membrane translocation. To test this hypothesis, proliferating hippocampal mouse (HT22) cells were transiently transfected with plasmids containing Munc13 DNA tagged with green fluorescent protein (GFP) and a membrane translocation assay was implemented. Activation of the protein was identified by accumulation of GFP fluorescence around the peripheral of the cell. Effects of Bryo were quantified and compared to those of a phorbol ester previously established to activate Munc13s, 12‐O‐Tetradecanoylphorbol‐13‐acetate (TPA). In this study, we demonstrate Bryo activation of over‐expressed GFP‐tagged Munc13‐1 and bMunc13‐2 in HT22 cells. Furthermore, by employing immunocytochemistry and immunoblotting of differentiated HT22 cells and primary hippocampal neurons, we observed increased expression of Munc13‐1. To conclude this project, we plan to continue studying the effects of Bryo on bMunc13‐2 activation and exploring the effects of Bryo treatments on the expression levels of Munc13 isoforms. Findings of this study suggest that Bryo's neuroprotective properties should be further studied in terms of Munc13 isoforms modulation along with those of PKC. The interaction between Munc13 isoforms and Bryo provides a novel molecular target that could be utilized to modulate neuronal synapses under dysfunctional synaptic conditions, like those present during the onset of AD. Support or Funding Information NIH R01 Grant (1R01AA022414‐01A1)