Epigenetic Regulation of Neural Development and its Use in Peripheral Nerve Regeneration
Leon J Nesti, MD, PhD1; Christopher Daniels, MD2; Youngmi Ji, PhD1; Stephen Fernicola, MD2; Edmund C Nesti, PhD3
1Uniformed Services University, Bethesda, MD, 2Walter Reed, Bethesda, MD, 3Alcamena Stem Cell Therapeutics, Spencerville, MD
BACKGROUND: Improving peripheral nerve regeneration and functional recovery after peripheral nerve injury remains a challenging clinical problem. We have previously found trauma-induced mesenchymal progenitor cells (MPCs) at these injury sites, and demonstrated their ability to secrete neurotrophic factors.
The repressor element-1 silencing transcription factor (REST) acts as a master regulator of neurogenesis by repressing terminal neuronal differentiation. Previous reports have found that REST was decreased following central nervous system insult, but the role that REST plays in peripheral nerve injury and the associated pathways are not well described.
C-terminal domain small phosphatase-1 (CTDSP-1) phosphorylates and stabilizes REST at a specific regulatory site. Using a novel peptidomimetic decoy to bind CTDSP-1 at this binding site limits its phosphorylation ability. Without the CTDSP-1 blockade on REST, neurogenic gene expression and increased neurotrophic factor expression can facilitate neuron axonal growth after nerve injury. It is not known if this peptidomimetic is able to access CTDSP-1 at its site of action, the nucleus.
HYPOTHESIS: We believe that the CTDSP-1 binding peptidomimetic will accumulate in the nucleus and modulate neurotrophic factor expression.
METHODS: MPCs were seeded onto coverslips on a 24-well plate. The next day, the cells were treated with the CTDSP-1-binding peptidomimetic, containing a FLAG-tag. Cells were harvested at 1 day after treatment, and fixed for immunocytochemistry with FLAG antibody for the peptidomimetic and Hoechst 33342 for the nucleus. Secondary antibodies 488 anti-mouse (as FLAG antibody) and 594 phalloidin (for cell morphology) were used for fluorescent visualization. Images were taken using confocal laser scanning microscopy. REST expression after peptide treatment was analyzed by Western blot, using a wild-type / mutant construct.
RESULTS: The fluorescent tag for the CTDSP-1-binding peptidomimetic was collocated with fluorescent stain that binds DNA. That is, the peptidomimetic gained access to the nucleus in trauma-induced mesenchymal progenitor cells. In Figure 1, panel B demonstrates the FLAG antibody, associated with the peptide. Panel C shows the Hoechst staining of the nucleus, and panel D demonstrates the colocalization. Western blot analysis demonstrated decreased fluctuation of REST expression after peptide treatment in the mutant-REST cells compared to a 7.8% decease seen for the wild-type.
SUMMARY:
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