A Novel Approach to Peripheral Nerve Regeneration: Epigenetic Reprogramming via Targeting of Carboxy-terminal Domain Small Phosphatase 1 (CTDSP1)
Noreen M. Gervasi, PhD1, Alexander Dimtchev, MD PhD1, Marvin E Dingle, MD2, DesRaj M. Clark, MD2, Andres S. Piscoya, MD2, Robert M Putko, MD2, Michael David Bedrin, MD2, Alexander Pisarchik, PhD3 and Leon J. Nesti, MD, PhD4, (1)Uniformed Services University, Bethesda, MD, (2)Walter Reed National Military Medical Center, Bethesda, MD, (3)Alcamena Stem Cell Therapeutics, Baltimore, MD, (4)Orthopaedic Surgery, Walter Reed National Military Medical Center, Bethesda, MD
Introduction: There are major clinical and economic consequences of Peripheral Nerve Injury (PNI) in our society. While peripheral neurons do have an inherent ability to regenerate axons in response to injury, full functional recovery is not always achieved and motor/sensory dysfunction, as well as chronic pain are common. Transcriptional changes in both injured neurons and adjacent support cells are necessary for successful regeneration and target organ reinnervation. Repressor Element 1 Silencing Transcription factor (REST), a master transcriptional regulator of neuronal genes, represses the expression of genes required for axon regeneration and synapse formation. Sustained upregulation of REST after nerve-injury inhibits regeneration and drives the development of chronic pain. Carboxy-terminal domain small phosphatase 1 (CTDSP1) prevents REST targeting to the proteasome therefore stabilizing REST. Knockdown or inhibition of CTDSP1 would in theory promote axon regeneration. This study explores the effects of CTDSP1 knockdown on axon regeneration, as well as on the expression of neurotrophic factors in both support cells present at the site of injury - such as mesenchymal progenitor cells (MPCs) - and in dorsal root ganglion (DRG) neurons. In addition, a newly developed CTDSP1 inhibitor is tested.
Materials and Methods: Cultured human MPCs or rat DRG neurons were transfected with REST or CTDSP1 specific siRNA or incubated with a CTDSP1 inhibitor. Brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) expression was analyzed by RT-qPCR and Western Blot. BDNF in cell culture medium was quantified by ELISA. Axon regeneration was quantified measuring the length of the longest neurite of a neuron.
Results: Knockdown of REST or CTDSP1 in MPCs resulted in increased expression of BDNF and NGF. Similar results were obtained with a CTDSP1 inhibitor. In addition, knockdown of CTDSP1 led to increased release of BDNF in cell culture medium from MPCs and to reduced levels of REST protein. Finally, knockdown of CTDSP1 in DRG neurons resulted in increased levels of BDNF and increased DRG neurite growth rate.
Conclusions: Preventing CTDSP1-mediated stabilization of REST via CTDSP1 knockdown or a CTDSP1 inhibitor promotes expression of neurotrophic factors in both DRG neurons and MPC support cells, and boosts DRG neuron regeneration. Therapeutic targeting of CTDSP1 activity may therefore represent a novel epigenetic strategy to promote peripheral nerve regeneration after PNI.
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