Trehalose Activates CRE-Dependent Transcriptional Signaling in HT22 Mouse Hippocampal Neuronal Cells: A Central Role for PKA Without cAMP Elevation

Abstract

Cyclic adenosine 3′,5′monophosphate (cAMP) regulated element binding protein (CREB) is a transcription factor involved in many different signaling processes including memory storage and retrieval. The mouse hippocampal neuronal cell line HT22 is widely used as a model system for neuronal cell death and cellular signal pathway investigations. For the present work a variant of HT22 with a stably expressed CRE-luciferase (CRE-luc) reporter (HT22CRE) is introduced, characterized and used to investigate cAMP-dependent and independent CRE-dependent signal processes. Trehalose (Mykose or 1-α-Glucopyranosyl-1-α-glucopyranosid) is a naturally occurring disaccharide consisting of two α,α′,1,1-glycosidic connected glucose molecules in a wide range of organisms but usually not found in mammals. Trehalose has been shown to activate autophagy, a process which regulates the degradation and recycling of proteins and organelles. The exact processes how trehalose application works on mammalian neuronal cells is not yet understood. The present work shows that trehalose application dose-dependently elevates CRE-luc activity in HT22 cells and acts synergistically with cAMP-elevating agents. In this pathway cAMP-dependent protein kinase (PKA) appears to be the most important factor and the stress kinase p38 and protein tyrosine kinases (PTKs) act as modulators.

Influence of Phosphodiesterase Inhibition on CRE- and EGR1-Dependent Transcription in a Mouse Hippocampal Cell Line

“…It can be expected that, at lower doses, the inhibition by Rp -8Br-cAMPS co-application will be even higher. The effect of 100 µM IBMX, for example, was reduced by 80% by Rp -8Br-cAMPS (500 µM), similar to what was previously shown for forskolin (1 µM) in HT22CREluc cells [ 14 ]. Here, we chose effect-saturating concentrations of IBMX, forskolin, and Sp -5,6-DCl-cBIMPS to compare the different PKA-activating agents and principles for the purpose of direct comparison.…”

“…Considering single time-point experiments, these differences may influence results. The last point had already been described before in HT22CREluc cells [ 14 ] and was confirmed here for HT22EGR1 cells and PDE inhibitors. This may be relevant for some types of experiments like those presented here.…”

“…HT22CREluc cells were produced as described [ 14 , 32 , 33 ] In short, HT22EGR1luc cells were made following the same procedure described for HT22CRE, but using an EGR1 reporter plasmid kindly provided by Dr. Pavel Krejici [ 15 ] Cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, Deisenhofen, Germany) with 10% fetal bovine serum (FBS), penicillin/streptomycin (100 U/mL) and GlutaMax (Life Technologies, Darmstadt, Germany). Clones showing stable induction of CREluc or EGR1luc activity (more than 4-fold elevated RLU after application to 1 µM forskolin in comparison to untreated culture) were expanded and frozen at a density of 1.5 million cells/mL/vial at −80 °C in a freezing medium (ibidi GmbH, Planegg, Germany).…”

“…FGF1 [ 30 ] (Sigma, Deisenhofen, Germany) was stored in H 2 O/50% glycerin at −20 °C and dissolved in water or cell culture medium for the experiments. Luciferin [ 14 ] (Promega, Heidelberg, Germany) was dissolved directly in the cell culture medium at the indicated concentrations. Reagents or appropriate vehicles were applied to the media for the indicated periods.…”

“…For a closer pharmacological characterization of the impact of PDE inhibition on cAMP-dependent transcriptional signaling in mouse hippocampal neuronal cells, we constructed two immortalized reporter cell lines [ 14 ], stably transfected with either a cAMP-regulated element (CRE) or an “early growth response 1” (EGR1) luciferase reporter plasmid [ 14 , 15 ]. We used these cell lines (HT22CREluc and HT22EGR1luc) for the investigation of dose- and time-dependency of the response toward different PDE inhibitors.…”