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It was found in Chapter 5 that PH3S10 in the dorsal horn was reduced after depletion of descending serotonergic fibres from the RVM. 5,7-DHT-induced depletion of descending 5-HT has been shown to lead to a reduction in spinal formalin-induced PERK, Zif268, and PMeCP2 (Géranton et al., 2008; Svensson et al., 2006) and has implicated descending 5-HT as having an overall pronociceptive role onto dorsal horn activity. Taking these results into consideration, a reduction in PH3S10 further suggested a key role in nociception.

7.2.4 Intracellular regulation of PH3S10 in nociceptive processing

Crucially, experiments from Chapter 6 proved a role for MSK1 in nociception, and suggested that MSK1 may act via PH3S10 to modulate the development of central sensitisation, reflected by attenuation of nocifensive behaviour in the second phase of the formalin response (Fischer et al., 2014), following i.t. inhibition by SB747651A. The

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downstream of PERK, and secondly showed that inhibition of MSK1 led to a reduction in both PH3S10 expression and nociceptive behaviour caused by formalin.

Although it is a well-known fact that ERK regulates nociceptive behaviour, ERK is known to have multiple downstream targets. Importantly, the experiments in Chapter 6 managed to dissect out one of the downstream ERK/MAPK signalling cascades which significantly contribute to nociception, particularly via inhibition of MSK1.

Similar to PH3S10 (and as mentioned in 7.2.2), the PMSK1 population was limited in size compared to PERK when investigated at 30 minutes. The PERK population was considerably larger than PMSK1, and there were many PERK positive cells that did not coexpress PMSK1 (Chapter 6, Experiment 1) suggesting a refinement of the signalling cascade at this particular step. However, experimental limitations regarding the time point investigating overlap should be noted, and other downstream targets of ERK not investigated here obviously cannot be ignored (i.e. Elk, Myc, RSK2).

The expression of PERK in dorsal horn neurons does not necessarily indicate outcomes of activity-induced gene transcription, as PERK is also involved in many early activity-induced intracellular events such as post-translational modification of receptors (Slack et al., 2004; Zou et al., 2000). It is not known if there are specific factors determining whether extracellular stimulation is sufficient to induce lasting changes in gene transcription and protein synthesis post-synaptically. However, MSK1 is the only kinase in the ERK/MAPK cascade that appears solely in the nucleus, and its positioning downstream of ERK could indicate that PMSK1 expression represents primary afferent activation of intracellular signalling sufficient to drive long-term nuclear changes contributing to nociceptive behaviour, and therefore potentially the development of chronic pain. The suggestion that PMSK1 may indicate a lasting outcome/consequence of extracellular activity is a striking one, as other studies have implicated MSK1 as having a key role in BDNF/MAPK signaling. Specifically, Correa et al (2012) suggest that MSK1 is the ‘apex’ of BDNF/MAPK signalling which is a molecular control determining the direction of neuronal output, and that MSK1 is a key homeostatic regulator of neural activity, implications which will be discussed in 7.4. Surprisingly, Chapter 6 results showed that MSK1 inhibition did not lead to a reduction in c-Fos expression in the dorsal horn. However, only a small reduction was expected, and it is entirely possible that IHC was insufficient at detecting a small reduction in c- Fos following MSK1 inhibition with SB747651A. A more sensitive technique such as ChIP might have indicated a change in PH3S10 mediated c-Fos transcription after formalin stimulation. ChIP experiments in other studies have proven that there is a

functional link between PH3S10 and the c-Fos expression, in particular cocaine- induced enrichment of PH3S10 at the c-Fos promoter has been observed in the brain (Brami-Cherrier et al., 2009). Investigating PH3S10-c-Fos associations using ChIP would be an important study contributing to the results in Chapter 6; it would also provide an important insight into other gene targets partially mediated by PH3S10 activity. Please see 7.5 Future directions.

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Figure 7.3 Summary of main findings in this thesis Box A) Expression of molecular targets investigated in the dorsal horn under naïve, injured, and injured-5-

HT-ablated conditions. Red box indicates area of investigation. Box B) Percentage colocalisation of formalin-induced PH3S10 population with markers of the pain pathways. Box C) Pharmacological inhibition of ERK/MAPK signalling by SL327 led to a reduction in formalin-induced PERK, PMSK1, and PH3S10. In a

separate experiment, inhibition of MSK1 by SB747651A reduced PH3S10 and prevented the full expression of pain states. Horizontal arrows indicate no change in injury-induced expression of molecular targets following drug treatment. Black inhibitory symbols indicate reduction in injury- induced expression of target following 5,7-DHT ablation or drug treatment.

7.3 MSK1: a key signalling molecule governing the post- synaptic

structural plasticity underlying hyperalgesia and allodynia

The induction and maintenance of central sensitisation requires a number of structural and functional changes post-synaptically, which can be separated into short-term post- translational events, such as receptor subunit phosphorylation or trafficking (Carvalho et al., 2000; Chen and Roche, 2007; Lau and Zukin, 2007), as well as longer-term events which require gene transcription and synthesis of new protein, the same way that LTP can be distinguished by early- and late- LTP phases. Considering the importance of intracellular signalling in activity-dependent neural function, there appears to be a clear functional distinction between early signalling events responsible for immediate post-translational consequences of Ca2+ entry, such as PKA, PKC, and CaMKII signalling, which are known to converge upon ERK (Hu et al., 2003; Ji et al., 2009), and further downstream signalling inclusive of ERK and MSK1. ERK and MSK1 therefore integrate a number of signalling events, regulating the transduction of cell surface-to-nucleus communication and thus long-term translational consequences of plasticity (Kelleher et al., 2004; Kelleher et al., 2004).

The findings in this thesis suggest that MSK1 could be a regulator of the long-term events involved in central sensitisation. Studies have shown that at early time points after acute inflammation (Fang et al., 2002; Fang et al., 2003; Fang et al., 2003; Larsson and Broman, 2008), phosphorylation of the AMPAR GluR1 subunit is increased, indicating a preferential switch from GluR2 to GluR1 subunit expression, which results in Ca2+ permeability of AMPAR. The preferential expression for calcium permeable GluR1 AMPAR subunits has also been observed at later time points (3d) of longer-term persistent pain states induced by i.pl. CFA (Vikman et al., 2008) which suggests a role for MSK1 regulating gene expression and translation of the GluR1 subunit. As previously discussed (Chapter 6), MSK1 has been shown to regulate the cell surface expression of AMPAR GluR1 and dendritic spine volume in the hippocampus, which has been suggested to occur via regulation of downstream target

Arc/Arg3.1, a CREB-mediated IEG (Bramham et al., 2008; Chowdhury et al., 2006;

Correa et al., 2012; Shepherd and Bear, 2011). Notably, it has been shown that Arc/Arg3.1 is acutely upregulated in the dorsal horn following inflammation or i.t. delivery of BDNF, and 70% of these cells were found to express enkephalin. Furthermore, Arc/Arg3.1 was observed in a small percentage of NK1 positive neurons (Hossaini et al., 2010).

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2007 OTHERS). Studies have shown that CREB is, in fact, an effector of central sensitisation (Latremoliere and Woolf, 2009). CREB is phosphorylated by a variety of signalling events in the dorsal horn post-injury, and leads to the transcription of several genes including c-Fos, NK1, and TrkB (Ji et al., 2009; Kawasaki et al., 2004). Although the interpretations for the role of MSK1 are speculative, the evidence likely points to the fact that MSK1 is a key signalling molecule mediating the events occurring at the cell surface down to the nuclear level to regulate gene expression.