Date of Award

1-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Science

First Advisor

Rasheed Al Hammadi

Second Advisor

Safa Shehab

Third Advisor

Eric Brown

Abstract

Death-associated protein kinase 1 (DAPK1) is a calcium/calmodulin (Ca2+/CaM) regulated serine/threonine kinase. An increasing body of evidence supports the significance of DAPK 1 protein in cancer and CNS diseases. The role of DAPK in peripheral nerve regeneration and neuropathic pain remains completely unexplored. The aim of this study is to investigate the expression pattern of DAPK1 along with key pro-and anti-apoptotic cell signaling molecules (p53, Bax, Akt, ERK5, p38) and to verify the possibilities of DAPKI1-NMDA NR2B relationship in dorsal root ganglion neurons (DRG) after 2 hours, 7 days and 14 days following a sciatic nerve injury. Gene expression analysis and immunohistochemistry were used to assess the effects of nerve injury. ATF3 was used as a neuronal injury marker. The results showed that DAPK1 mRNA was expressed and translated to functional protein in normal DRG neurons. Soon after a sciatic nerve injury (2 hours), DAPK1 was significantly up-regulated (p<0.05, 2.2 fold) in the injured L4 and L5 DRG compared with the contralateral uninjured side. However, 7days after axotomy a profound decrease was observed in the DAPK1 level, with a further reduction that reached its minimum level at 14 days postoperatively. In addition, 7 days after injury, most of the DAPK1 positive injured neurons (76.11%) were ATF3 positive, while after 14 days DAPK1 immunoreactivity significantly decreased (53.89%) in injured ATF3 positive neurons. Interestingly, DAPK1, p53 and Bax exhibited a similar expression pattern in axotomized lumbar DRG. The results also revealed that a sciatic nerve injury had no effects on the gene expression of ERK5, P38 and AKT at every time point. Moreover, NMDA NR2B mRNA expression increased after 7 days and continued to up-regulate significantly until 14 days postoperatively (p<0.05,3.6 fold). In contrast, our immunofluorescence results showed a decrease in the protein level in DRG neurons during this time period; but a strong positive NMDA NR2B immunoreactivity appeared in the satellite cells that surround the injured large-sized neurons in L4 and L5 DRG neurons. In addition, immunofluorescence double labelling revealed that DAPK1 and NMDA NR2B are co-localized in normal and injured DRG neurons. In conclusion, the down-regulations of DAPK1 following sciatic nerve injury, along with other vital pro-apoptotic players promoting neuronal survival might shed light on the mechanisms of peripheral nerve regeneration. The results also suggest that NMDA NR2B might modulate neuropathic pain through satellite cells, and not through neurons, 7 and 14 days after peripheral nerve injury.

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