RNA-driven Epigenetic Modulation of Neurotropic Factors Through CTDSP1 and REST Pathway As a Novel Strategy in Peripheral Nerve Regeneration After Traumatic Injury
Noreen M. Gervasi, PhD1,2, Marvin E Dingle, MD3, DesRaj M. Clark, MD3, Andres S. Piscoya, MD3, Alexander Dimtchev, MD PhD1,2 and Leon J Nesti, MD PhD1,3, (1)Uniformed Services University, Bethesda, MD, (2)Henry M Jackson Foundation, Bethesda, MD, (3)Walter Reed National Military Medical Center, Bethesda, MD
Modulation of CTDSP1 activity in mesenchymal progenitor cells as a novel strategy to promote peripheral nerve regeneration after traumatic injury
Introduction: Peripheral nerve injury is a frequent complication in high-energy musculoskeletal trauma. Current therapeutic interventions are hindered by limited regeneration of injured nerves and rarely lead to complete functional recovery. The regenerative ability of nerves depends in part on trophic support received from surrounding mesenchymal progenitor cells (MPCs) at the zone of injury. We have shown that MPCs that concentrate in traumatized tissue produce neurotrophic factors, which support neurite growth. The expression of neurotrophins such as BDNF, NGF and NT3, is regulated by repressor element-1 silencing transcription factor (REST), which is protected from degradation by C-terminal domain small phosphatase 1 (CTDSP1). While REST levels increase after central nervous system injury, the response in peripheral nerve injury is unclear. Here, expression profiles of REST, CTDSP1 and neurotrophins in traumatized muscle tissue and in MPCs are characterized, and effects of CTDSP1 knockdown on expression of neurotrophins in traumatized-tissue derived MPCs are examined as a potential way to promote peripheral nerve regeneration.
Methods: RNA and protein were isolated from traumatized muscle tissue and from traumatized muscle-derived MPCs at different time points post-injury. The expression of REST, CTDSP1 and neurotrophins were quantified by RT-PCR and Western Blot. MPCs were transfected with CTDSP1-targeting siRNA or non-targeting siRNA and cultured in either standard medium or neuroinduction medium. The effects of CTDSP1 knockdown on the expression levels of REST and neurotrophins were analyzed by RT-PCR. The levels of secreted BDNF were measured in cell culture supernatants by ELISA.
Results: REST, CTDSP1 and neurotrophin expression levels in traumatized tissue are altered in a time-dependent manner following traumatic injury. An inverse correlation between levels of CTDSP1 and neurotrophins was observed. Knockdown of CTDSP1 in MPCs resulted in higher expression levels of neurotrophins and increased secretion of BDNF.
Conclusions: The observed inverse correlation between CTDSP1 and neurotrophin levels in traumatized muscle tissue is consistent with CTDSP1 stabilization of REST and subsequent repression of neuronal genes expression, with decreased CTDSP1 leading to increased neurotrophin expression. Accordingly, knockdown of CTDSP1 in MPCs results in increased expression of neurotrophins, such as BDNF, which have been shown to promote neurite sprouting. Controlling the activity of CTDSP1 at the site of injury may therefore represent a novel strategy to promote nerve regeneration by increasing the availability of neurotrophins forming a favorable environment.
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