selectivity toward H3Rs (Łażewska et al., 2006) has been evaluated on it is behavioral effects on MK801-induced memory deficits applying novel object recognition test (NORT) and assessing treated mice on short- and long-term memory (STM and LTM).
Also, since anxiety and motor activity could confound learning and memory’s
performance of tested animals (Sadek et al., 2016c) , the effects of E169 on locomotor activity and anxiety-like behaviors of the same animals following NORTs were
investigated in OFT. Moreover, the abrogative effects of H3R agonist RAMH on the E169- provided memory-enhancing effects in NORT were tested.
With reference to our research objective in evaluating the effect of E169 on STM and LTM in MK801-treated mice. Our results showed that the H3R antagonist E169 effectively counteracted the STM and LTM deteriorating effects induced by MK801, since it improved performance of tested mice in the NORT for both STM and LTM by increasing the exploration time for novel object, indicating the memory improving properties of H3R antagonist E169. Our observations agreed with previous results
obtained for H3R antagonist/inverse agonist ABT-239 in the same test model (Winters et al., 2008). In the latter study, the authors concluded that cortical and hippocampal
cholinergic neurotransmissions were incorporated in the effects observed for ABT-239 (Winters et al., 2008). Moreover, our results agreed with many other previous preclinical observations applying NORT in rats and mice and assessing numerous H3R antagonists e.g. thioperamide and clobenpropit (Giovannini et al., 1999); pitolisant (Ligneau et al., 2007); GSK189254 (Giannoni et al., 2010); SAR110894 (Griebel et al., 2012), all of them were found to enhance memory deficits induced following systemic injection of scopolamine or MK801.
Interestingly, the E169-provided benefits in NORT were completely reversed when mice were concurrently injected with the centrally acting H3R agonist RAMH.
The latter findings are consistent with a previous study in which RAMH nullified the memory-improving effects of the H3R antagonist ciproxifan on LTM in experimental rodents (Pascoli et al., 2009).
Furthermore, another goal of the current project was to examine whether the PI3K-AKT-GSK3β signaling pathway participated in the observed memory-enhancing
effects of E169 in MK801-treated amnesic mice. Akt activation is well known for regulating the phosphorylation of several signaling molecules, including GSK-3β (Kandel & Hay, 1999). There are many phosphorylation sites on Akt, and specifically the phosphorylation of Ser473 by autophosphorylation and unknown kinases is critical in the control of Akt (Alessi & Cohen, 1998; Scheid & Woodgett, 2003; Sutton &
Chandler, 2002).
Accordingly, hyperphosphorylation of pathogenic tau was found to increase synthesis of β-amyloid and memory deficit are all caused by unphosphorylated GSK-3β (Giacobini & Becker, 2007; Hooper et al., 2008; Jaworski et al., 2011). The GSK-3β effect was discovered to be higher in the brains of patients diagnosed with AD (Hooper et al., 2008; Hye et al., 2005; Leroy et al., 2007). Also, it has been suggested that H3R antagonists can enhance cognitive functions in a variety of cognitive disorders by increasing the release of ACh and inducing pGSK-3β (Bitner et al., 2011; Passani &
Blandina, 2011).
Analyses of the results observed in western blot assessments indicated that acute systemic treatment with H3R antagonist E169 resulted in a significant reduction of the protein overexpression of the cascade, and as witnessed as a significant reduction in the overexpression of PI3K, PAKT /AKT ratio, and PGSK-3β/GSK-3β ratio, and as
compared to the protein levels of hippocampal tissues of naïve MK801-treated mice. The latter results indicate that H3R antagonist E169 improved memory deficits induced by MK801 since it will reduce the overexpression of pGSK-3β.
In a further experiment, mice which were given RAMH failed to display the beneficial effects of E169 on phosphorylation of the GSK-3β. Indicating the abrogative effect of RAMH confirming that the memory-enhancing effect was due to H3R.
Our observations also showed that the phosphorylation of Ser473- Akt, and Ser9- GSK-3β in the hippocampus of mice was overactivated by the amnesic effects of
MK801, and these results agreed with previous preclinical experiments that were carried out in experimental rodents (Svenningsson et al., 2003). Surprisingly, (Kitagawa et al., 2002) found that only phosphorylation of Ser473-Akt was overactivated when rat cultured neurons were exposed to glutamate, but no overactivated phosphorylation of
Thr308-Akt was observed. However, and in disagreement with the latter results, our observations showed that the NMDA receptor antagonist MK801 was capable of over activating Ser473-Akt phosphorylation (Kitagawa et al., 2002). The latter contradiction could also be explained by a previous study in which NMDA dose-response curve for the stimulation of phospho-PKB (Akt) was found to be of a biphasic character (dose- dependent effect) (Sutton & Chandler, 2002).
Moreover, the effects of the PI3K inhibitor LY294002 on STM and LTM in MK801-treated mice were investigated. The results observed showed that LY294002 impaired performance in NORT in control mice, and the obtained impairments in our experiments were comparable to those reported in several previous preclinical studies in rodents (Barros et al., 2001; Chen et al., 2005; Slouzkey et al., 2013). Contrary to the results observed in control mice, the effects of LY294002 on MK801-treated mice were found to be of improving properties on performance of amnesic mice in NORT, and this could be explained with it is effect on counteracting the MK801-induced overexpression of pGSK-3β. The latter findings were confirmed with western blot analyses which revealed a significant reduction in the overexpression of PI3K, PAKT /AKT ratio and PGSK-3β /GSK-3β ratio, and as compared to MK801-treated and amnesic mice. These observations demonstrate that the PI3K inhibitor LY294002 positively affects memory in the MK801-induced amnesic mice due to it is balancing effect on PI3K /AKT /GSK- 3β pathway, since the overexpression of pGSK-3β induced by MK801 was significantly reduced.
In the OFT, our observations showed that locomotion and anxiety levels at administered doses are not affected, as time spent in the periphery, time spent in the center, and total distance travelled for mice injected with E169 (2.5-10 mg/kg) did not differ from control group. These results are of high significance since improved
performance in NORT can be the consequence of several variables not related to memory-enhancing effects of test compound E169, such as modifications in emotional responding or in spontaneous locomotor activity (Eissa et al., 2018). Consequently, the observed results in OFT exclude all these confounding factors. These results in OFT are, also, in agreement with recent previous observations in which acute systemic
administration of several H3R antagonists, e.g. DL77 (2.5–10 mg/kg) and E159 (2.5–10
mg/kg) failed to modify spontaneous locomotor activity of the same animal species in the OFT (Alachkar et al., 2017).