VALLE DE VILCABAMBA

The investigation into MSK1 was interesting in itself as a novel target never before explored within the context of nociception. Additionally, being upstream of PH3S10, MSK1 was an ideal target for interfering with PH3S10 expression in Experiment 2:

Does inhibition of MSK1 activity attenuate formalin behaviour and block formalin- induced PH3S10 and c-Fos expression?

In general, studies investigating the role of histone modifications in neuronal function and behaviour have pharmacologically targeted the enzymes responsible for either imparting or removing the histone marks. In particular, these studies have used HAT or HDAC inhibitors to examine the effects of manipulating histone acetylation levels in a relatively non-specific manner (Barrett and Wood, 2008; Gräff and Tsai, 2013; Peixoto and Abel, 2013). In order to manipulate PH3S10, there were two options: MSK1, the kinase responsible for PH3S10, or protein serine/threonine phosphatase 1 (PP1) the kinase responsible for H3S10 dephosphorylation (for discussion of PP1 refer to 6.4.3.5). In order to choose an appropriate target, we investigated the distribution of PMSK1 in the dorsal horn after noxious stimulation. The results in this chapter (Experiment 1) indicated that PMSK1 and PH3S10 populations had similar distributions within the superficial dorsal horn. The majority of PH3S10 colabelled with PMSK1 (86%), and conversely the majority of PMSK1 colabelled with PH3S10 (59%) (Figure 6.9 B). While the number of PH3S10 nuclei coexpressing PMSK1 was very high, the reverse figure for the PMSK1 population indicated a substantial but not complete amount of population overlap (59%). However, this again could have been an issue of timing as 30 minutes may not have been optimal for PMSK1 expression. Additionally, PH3S10 is not the only downstream target of MSK1, as MSK1 is also known to phosphorylate transcription factors CREB and activating transcription factor 1 (ATF1) in vitro (Arthur et al., 2004; Soloaga et al., 2003; Wiggin et al., 2002), so perhaps a complete colocalisation between PMSK1 and PH3S10 should not be expected. Nonetheless, the substantial overlap between PH3S10 and PMSK1 populations suggested that MSK1 would be a good target for interfering with PH3S10 expression.

Many studies have investigated the role of MSK1 using genetically modified mice (Brami-Cherrier et al., 2005; Chandramohan et al., 2008; Chwang et al., 2007; Correa et al., 2012; Karelina et al., 2012). Recently however, a new MSK1 inhibitor, SB747651A has become available (Tocris, UK). Prior to the release of SB747651A, studies attempting to pharmacologically target MSK either utilised upstream inhibition of MEK (using inhibitors PD98059, SL327, U0126), Ro 31-8220 or H89- the latter two being non-selective MSK1 inhibitors also known to inhibit PKC and PKA, respectively, as well as other kinases. In contrast, using SB747651A causes less interference with other cell signalling activity, and allows for a refinement of MSK1 inhibition. Currently, no published work has used SB747651A in vivo, but recent evidence has conclusively shown that it has improved selectivity of MSK1 compared to Ro 31-8220 and H89 in

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The use of global MSK1-/- mice has shown that MSK1 itself regulates a variety of forms of neural plasticity. In particular, MSK1 regulates memory formation (as shown by contextual fear conditioning, the forced swim test, and spatial and object- recognition tasks), environmental enrichment-induced cognitive enhancement, visual cortical plasticity, cocaine addiction, and neurodegeneration (Brami-Cherrier et al., 2005; Chandramohan et al., 2008; Chwang et al., 2007; Karelina et al., 2012; Putignano et al., 2007; Roze et al., 2007). MSK1 has also been shown to modulate dendritic morphology and synaptic plasticity involved in hippocampal environmental enrichment and homeostatic synaptic scaling, and is thought to do so by regulation of AMPA subunit GluR1 expression via the IEG arc/arg3.1, which is also known to be upregulated in the spinal dorsal horn by noxious stimulation (Correa et al., 2012; Hossaini et al., 2010).

Control experiments in this chapter indicated there were no upstream changes in PERK activation after i.t. delivery of SB747651A (10µM), which would support the targeted specificity of MSK1 inhibition within L4/5 of the superficial dorsal horn (Figure 6.12). However, it should be carefully noted that there was a trend toward a PERK reduction in the SB747651A-treated group, with only an n=4 (Figure 6.12, P=0.06). The significant reduction in formalin-induced PH3S10 observed following i.t. SB747651A (10µM) required an n=11/13 (Figure 6.10), a value much higher than the n=4 used in the control PERK experiment. Furthermore, i.t. SB747651A (10µM) caused a significant attenuation of flinching behaviour in the first phase of the formalin response (Figure 6.11); thus, if spinal PERK was directly indicative of nociceptor activity it would not be surprising to observe a significant reduction in PERK in SB747651A-treated animals with a higher n number.

Quantification of IHC indicated that there were also no changes in PMSK1 expression following SB747651A. However, this was expected- SB747651A is a specific inhibitor of the MSK1 N-terminal kinase, the domain which is reportedly essential for the phosphorylation of downstream MSK1 substrates (McCoy et al., 2005). The antibody used to detect PMSK1 was specific to phosphorylation at Threonine 581 in the C- terminal kinase domain, activity which precedes N-terminal activity. It is worth noting that SB747651A can inhibit ribosomal s6-kinase (RSK) and PKA at low levels (Naqvi et al., 2012). Non-specific inhibition of PKA could have potentially had an effect on the expression of PH3S10, as MEK is known to integrate upstream PKA and PKC activity in dorsal horn signalling (Hu et al., 2003). However, the fact that no changes in PERK were observed eliminates this potential confounding factor. RSK2 occurs downstream of ERK and therefore may be a factor requiring consideration.