Artemisinine br Experimental procedures br Results Hdc KO mi

Experimental procedures
Results Hdc-KO mice show basal activation of the MAPK and AKT/GSK3β pathways in the dorsal striatum (Rapanelli et al., 2014). These signaling pathways are differentially regulated by the H3 receptor in dMSNs and iMSNs in wild-type mice (Rapanelli et al., 2016). To better characterize their regulation with cell-type specificity in Hdc-KO mice, we crossed Hdc-KO mice with reporter mice in which dMSNs and iMSNs are labeled with distinct epitope tags (Bateup et al., 2008). We used triple-immunostaining for FLAG (the epitope tag that labels dMSNs), Myc (labeling iMSNs), and phospho-proteins implicated in MSN intracellular signaling, 30 min after systemic saline or RAMH challenge.
Discussion Alterations in the brain’s Artemisinine modulatory system have been implicated as a contributor to the development of tics and of TS by a series of genetic studies (Ercan-Sencicek et al., 2010, Fernandez et al., 2012, Karagiannidis et al., 2013). The Hdc knockout mouse recapitulates a rare but high-penetrance genetic cause of TS, inactivation of the histidine decarboxylase gene, and constitutes a promising model of tic pathophysiology (Ercan-Sencicek et al., 2010, Castellan Baldan et al., 2014, Pittenger, 2017). Hdc-KO mice exhibit repetitive behavioral pathology, DA dysregulation, altered histamine receptor levels, and elevated markers of cellular activity and intracellular signaling in the striatum (Dere et al., 2003, Castellan Baldan et al., 2014, Rapanelli et al., 2014, Rapanelli et al., 2017, Xu et al., 2015). H3R has historically been described as a presynaptic Gαi-coupled receptor; it can negatively regulate the release of HA itself as well as of DA, glutamate, and other neurotransmitters (Schlicker et al., 1994, Haas et al., 2008, Ellender et al., 2011). However, it is increasingly clear that H3R also exists postsynaptically, in which context its signaling properties are complex and are modulated by its interactions with other cellular components, including DA receptors (Ferrada et al., 2008, Ferrada et al., 2009, Moreno et al., 2011, Moreno et al., 2014). Indeed, postsynaptic signaling may be a primary function of the H3 receptor in the striatum (Panula and Nuutinen, 2013). Distinct H3R signaling pathways in dMSNs and iMSNs have been described ex vivo (Ferrada et al., 2009, Moreno et al., 2011). We have recently replicated and extended this work in vivo, finding that H3R regulates MAPK signaling in dMSNs and Akt/GSK3β signaling in iMSNs in WT mice (Rapanelli et al., 2016). In the current study, we set out to determine whether MAPK and Akt signaling are also differentially regulated by H3R in Hdc-KO mice. We have previously shown this receptor to be upregulated in the striatum of these KO animals, both at the level of mRNA expression and at the level of radioligand binding (Rapanelli et al., 2017). Several findings emerge. First, consistent with previous work (Ferrada et al., 2008, Ferrada et al., 2009, Moreno et al., 2011, Panula and Nuutinen, 2013, Moreno et al., 2014, Bolam and Ellender, 2015, Rapanelli et al., 2016), H3R signaling differentially affects intracellular signaling in dMSNs and iMSNs. This supports the conclusion that H3R interactions with other receptors, most prominently the D1R and D2R dopamine receptors, critically modulate its postsynaptic signaling properties. The one previously described effect of RAMH that we did not replicate here was the increased phosphorylation of Akt1 seen in dMSNs at an earlier time point (Rapanelli et al., 2016). This intriguing effect is transient – it is present 15 min after RAMH challenge but completely resolved by 45 min. It is likely that Akt regulation by RAMH in dMSNs is already resolved at the intermediate time point used in the current experiments (30 min after RAMH challenge). Second, baseline signaling dysregulation in Hdc-KO MSNs differs in dMSNs and iMSNs. We have previously reported alterations in MAPK, rpS6, and Akt signaling in this knockout (Rapanelli et al., 2014). The current study adds nuance by dissociating effects in dMSNs and iMSNs. All abnormalities reported previously in undifferentiated striatum are replicated in the cell-specific effects here. Additionally, subtle alterations in rpS6 phosphorylation at S240/244 are seen in both cell types, but in opposite directions: basal phosphorylation of this site is reduced in Hdc-KO dMSNs and increased in iMSNs (only the latter effect reached statistical significance). This was not observed in undifferentiated striatum (Rapanelli et al., 2014), perhaps because the two effects obscure each other in when dMSNs and iMSNs are not examined separately. This emphasizes the utility of employing tools that allow separate quantification of signaling in the two MSN types.