A novel non-viral knockdown strategy targeting retinoblastoma protein for regeneration of peripheral axons

• Author / Creator

  Zubkow, Kasia

• Peripheral nerve degeneration as a result of traumatic nerve injury is a common neurological condition that is characterized by varying degrees of sensory, autonomic, and motor dysfunction. Due to the high prevalence of peripheral nerve damage, there is a pressing need to develop novel therapies that augment the growth capacity of peripheral nerves in order to achieve optimal functional recovery. Tumor suppressor proteins are a class of anti-proliferative molecules that primarily function to hinder cancer development, but whose experimental suppression has been correlated with increased levels of peripheral axon regeneration. Retinoblastoma protein (Rb1) is one such tumor suppressor, whose role in the regeneration of peripheral nerves has yet to be fully elucidated. Rb1, by binding to the transcription factor E2F1, impedes its ability to promote the production of proteins necessary for cell cycle progression (Harbour et al., 1999). By inhibiting the action of Rb1 via transfection with small-interfering RNAs (siRNAs), our lab has previously highlighted Rb1 as a promising new molecular target for enhancing axon outgrowth (Christie et al., 2014).
  Through the use of a novel, non-viral siRNA transfection technique, the current study sought to investigate the impact of a delayed knockdown of Rb1 after injury on the regeneration of distal sensory axons in the skin. Here we show that repeated injections of siRNA into the mouse hind paw followed by electroporation is capable of producing a partial knockdown of Rb1 mRNA in the ipsilateral lumbar dorsal root ganglia of intact, healthy mice, as well as those that had previously undergone a sciatic nerve injury. In order to assess the effects of a partial, delayed knockdown of Rb1 on the regeneration of epidermal sensory axons and functional recovery after nerve injury, we applied the aforementioned transfection technique in the weeks following a sciatic nerve crush and subsequently evaluated regenerative and functional outcomes at multiple time points through behavioural and electrophysiological testing, and histological indices.
  Immunohistochemical analysis of the epidermis in footpad biopsies harvested 28 days post-crush showed that mice treated with Rb1-targeted siRNA possessed a significantly greater number of axon profiles crossing the dermal-epidermal junction than did those treated with scrambled control siRNA sequences. However, when innervation levels were examined at day 40, this pro-growth effect in the Rb1 siRNA-treated cohort was lost, indicating that the regenerative benefits observed on day 28 did not endure over an extended timeline. These histological findings were mirrored by the behavioural tests of thermal and mechanical sensitivity as well as by the electrophysiological measures of nerve conduction, which all lacked substantial between-group differences between the Rb1 siRNA and scrambled siRNA-treated cohorts, which would have indicated an improvement in functional recovery following Rb1 knockdown.
  Altogether, the findings in this study suggest that hind paw injections of siRNA coupled with electroporation represents a novel and less-invasive means of procuring a partial genetic knockdown in sensory neuron cell bodies. We also conclude that although a delayed knockdown Rb1 improves the ability of regenerating axons to reinnervate the epidermis following injury, these early benefits do not persist at later stages beyond the point of siRNA treatment cessation. Taken in conjunction with previous findings reported by Christie et al. (2014), this suggests that there is an optimal time during which Rb1 suppression is most advantageous for promoting regrowth after injury. Knowledge of how the timing of molecular interventions affects regenerative outcome is clinically relevant and may aid in the development of optimized protocols for pharmacological intervention or gene therapy that could accompany existing surgical techniques for improved patient recovery after peripheral nerve trauma.

• Subjects / Keywords

  • peripheral nerve injury
  • regeneration
  • retinoblastoma protein
  • RNAi

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3F47H943

• License

  Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.