The Effect of GDNF on Schwann Cells and their Role in Peripheral Nerve Regeneration
Date of Award
Doctor of Philosophy (PhD)
Peripheral nerve damage occurs in 1-3% of all traumatic injuries and results in ~200,000 surgical interventions performed each year. Unfortunately, only 25% of surgical patients regain full motor function and less than 3% regain sensation. The gold standard surgical repair method for long gaps is the autologous nerve graft, or autograft; however, significant drawbacks include: donor site morbidity, risk of infection, and increased cost. Acellular nerve allografts (ANAs), nerves harvested from cadaveric donors, have shown promise in improving regeneration. However, as ANAs must undergo a decellularization process to remove immunogenic material, they lack much of the trophic support necessary to encourage regeneration of axons to distal targets. The main source of growth factor support and axonal guidance cues comes from Schwann cells (SCs), the glial cells of the peripheral nervous system. Therefore, SC transplantation and growth factor delivery may improve ANA outcomes. In addition to their role in regeneration, SCs have also been classified into motor or sensory phenotypes based on their association with either motor or sensory axons, which become dysregulated when removed from axonal contact. Understanding these phenotypes may play an important role in targeting motor neuron regeneration and functional motor recovery. Furthermore, the use of glial-cell line derived neurotrophic factor (GDNF), a potent stimulator of axon regeneration, has been shown to affect SC differentiation and drive SCs into native motor or sensory phenotypes. Therefore, in this dissertation, we studied the effect of GDNF on SC phenotype and the resulting interactions with neurons in vitro. In addition, we developed a spatially and temporally controlled GDNF delivery system using lentiviral (LV)-based transduction of SCs in order to improve nerve regeneration in a critical rat sciatic nerve injury model.
First, we explored the interaction between phenotypically matched/mismatched SCs and neurons using a microdevice platform. Our results indicate that despite loss of axon contact for a significant period, SCs are capable of promoting neurite growth in a phenotype specific manner. Interestingly, preconditioning of SCs with GDNF can overcome phenotype mismatch. To understand this better, we examined GDNF mRNA and protein levels and found that preconditioning of SCs with exogenous GDNF leads to increased endogenous levels. Next, we studied the mechanism by which GDNF drives SC maturation and differentiation. Through targeted siRNA knockdown, we found that a Fyn-mediated GDNF pathway drives SC maturation and endogenous GDNF production. Furthermore, by disrupting this pathway, we remove any positive effects GDNF has on mismatched SC-neuron cultures. We also found that sustained delivery of GDNF using a heparin-based delivery system (HBDS) is capable of driving SC maturation and differentiation. Finally, we designed a GDNF delivery platform using both the HBDS and LV-transduced SCs expressing GDNF under tetracycline induction. The timing of GDNF over-expression was studied in order to promote enhanced regeneration and prevent axons becoming entrapped in areas of high GDNF production. SCs were transduced with a tetracycline inducible GDNF (Tet-on GDNF) LV vector and transplanted into distal nerve stumps, while 3 cm GDNF HBDS-modified ANAs were used to bridge the critical nerve gap. We found that 6 weeks of GDNF over-expression from SCs led to improved axonal regeneration and muscle mass recovery. Our results have shown that GDNF has a potent effect on SCs and axons and may have a significant contribution toward designing therapies for peripheral nerve injury.
Shelly E Sakiyama-Elbert
Donald Elbert, Kelly Monk, Kristen Naegle