This thesis examines the role played by small RNAs, in particular micro RNAs (miRNAs), in peripheral sensory neurons and their effects on regulating the neural transcriptome. The principal approach to investigating small RNAs was to ablate the RNAse III family endoribonuclease dicer. Dicer is a critical enzyme responsible for the maturation and functional activity of many species of small RNAs, such as miRNAs, and short interfering RNAs (Kim, Han & Siomi, 2009). In an earlier study using the Cre-lox P system in mice, dicer was conditionally ablated from the small diameter Nav1.8 expressing sensory neurons. This generated mice with otherwise normal developmental characteristics but a phenotype of reduced sensitivity to cold stimuli. Furthermore, following inflammatory insult, these animals displayed significant analgesia in comparison to their Cre negative littermate controls (Zhao et al., 2010). In this thesis, the role of dicer in all sensory neurons was investigated. Initially this was carried out using a Cre under the control of the Advil gene (Minett et al. 2012). This Cre is expressed in all sensory neurons and ablation of dicer using this Cre proved fatal. Dicer is therefore essential in early neural development and survival since dicer loss at the embryonic stage is fatal. In order to circumvent the fatal developmental loss of dicer, a newly developed Cre under the control of the Advil gene but conjugated to a mutated human oestrogen receptor ERT2 was employed (Lau et al., 2011). This allowed for normal adult development of the mouse and Cre mediated gene ablation only following the administration of the ERT2 agonist tamoxifen. Pan sensory neuron loss of dicer in adult mice had no detrimental effect on overall survival, or survival of the dorsal root ganglion (DRG) neurons, and was not associated with abnormal responses to acute thermal and mechanical stimuli. Following inflammatory and neuropathic insults, no behavioural differences were detected between dicer deficient or wildtype mice. In a study of the DRG transcriptome following pan sensory neuron dicer ablation using microarray and quantitive real-time reverse-transcription PCR (qRT-PCR), many genes associated with sensory neurons were down-regulated. The loss of neuron specific transcripts associated with the ablation of dicer is paradoxical since dicer products are canonically considered to negatively regulate gene expression. Further mining of the microarray data showed that a suppression factor responsible for silencing neuronal genes in non-neuronal cells termed the neuron restrictive silencing factor (NRSF) was significantly up-regulated in the dicer ablated DRG sensory neurons. Associated with this was a significant increase in its essential co-factors, small C-terminal domain phosphatase (Scp1). It was proposed that Dicer ablation in sensory neurons lead to an increase in NRSF and Scp1 production, which ultimately silences the expression of neuronal genes. In order to test this, neuronal genes that are understood to be regulated by NRSF such as Nav1.8 and Edg7 were investigated following miRNA transfection into dicer ablated DRG neurons that specifically target NRSF and Scp1. Furthermore, NRSF sequestration in dicerablated neurons suggested an enhancement of Nav1.8 expression. However, these experiments met with some technical challenges. This is the first study investigating the loss of dicer in the adult sensory neuron. The behavioural differences between the Nav1.8-Cre conditional embryonic dicer knockout and the Advillin-CreERT2 conditional adult dicer knockout suggest that dicer plays a critical role in the developing neuron but not the adult neuron. This therefore suggests that pharmacological modulation of dicer activity has little therapeutic value as a means to treat pain.
|Creators||Shepherd, S. T.|
|Publisher||University College London (University of London)|
|Source Sets||Ethos UK|
|Type||Electronic Thesis or Dissertation|
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