International Journal of Molecular Sciences Article Spatial Memory Training Counteracts Hippocampal GIRK Channel Decrease in the Transgenic APPSw,Ind J9 Alzheimer’s Disease Mouse Model Sara Temprano-Carazo 1,† , Ana Contreras 1,† , Carlos A. Saura 2,3 , Juan D. Navarro-López 1, *,‡ and Lydia Jiménez-Díaz 1, *,‡ 1 Neurophysiology & Behavior Lab, Centro Regional de Investigaciones Biomédicas (CRIB), School of Medicine of Ciudad Real, University of Castilla-La Mancha, 13079 Ciudad Real, Spain 2 Institut de Neurociències, Department de Bioquímica i Biología Molecular, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 3 Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CIBERNED), 28029 Madrid, Spain * Correspondence: juan.navarro@uclm.es (J.D.N.-L.); lydia.jimenez@uclm.es (L.J.-D.) † These authors share first authorship. ‡ These authors contributed equally to this work and share last authorship. Abstract: G-protein-gated inwardly rectifying potassium (GIRK) channels are critical determinants of neuronal excitability. They have been proposed as potential targets to restore excitatory/inhibitory balance in acute amyloidosis models, where hyperexcitability is a hallmark. However, the role of GIRK signaling in transgenic mice models of Alzheimer’s disease (AD) is largely unknown. Here, we study whether progressive amyloid-β (Aβ) accumulation in the hippocampus during aging alters GIRK channel expression in mutant β-amyloid precursor protein (APPSw,Ind J9) transgenic AD Citation: Temprano-Carazo, S.; mice. Additionally, we examine the impact of spatial memory training in a hippocampal-dependent Contreras, A.; Saura, C.A.; Navarro-López, J.D.; Jiménez-Díaz, L. task, on protein expression of GIRK subunits and Regulator of G-protein signaling 7 (RGS7) in the Spatial Memory Training Counteracts hippocampus of APPSw,Ind J9 mice. Firstly, we found a reduction in GIRK2 expression (the main Hippocampal GIRK Channel neuronal GIRK channels subunit) in the hippocampus of 6-month-old APPSw,Ind J9 mice. Moreover, Decrease in the Transgenic APPSw,Ind we found an aging effect on GIRK2 and GIRK3 subunits in both wild type (WT) and APPSw,Ind J9 J9 Alzheimer’s Disease Mouse Model. mice. Finally, when 6-month-old animals were challenged to a spatial memory training, GIRK2 Int. J. Mol. Sci. 2022, 23, 13444. expression in the APPSw,Ind J9 mice were normalized to WT levels. Together, our results support https://doi.org/10.3390/ the evidence that GIRK2 could account for the excitatory/inhibitory neurotransmission imbalance ijms232113444 found in AD models, and training in a cognitive hippocampal dependent task may have therapeutic Academic Editor: Cesar Borlongan benefits of reversing this effect and lessen early AD deficits. Received: 5 October 2022 Keywords: hippocampus; GIRK; Alzheimer’s disease; APPSw,Ind J9; spatial memory; excitatory/ Accepted: 1 November 2022 inhibitory imbalance Published: 3 November 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Over 55 million people worldwide have been diagnosed with dementia, of which 60–70% suffer from Alzheimer’s disease (AD) [1]. AD is characterized by extracellular accumulation of β-amyloid (Aβ) plaques and intracellular neurofibrillary tangles of hyper- Copyright: © 2022 by the authors. phosphorylated tau, leading to a progressive neurodegeneration and memory loss. Despite Licensee MDPI, Basel, Switzerland. the extended research in the topic, no current therapeutic disease-modifying intervention This article is an open access article exists (for a review, see [2]). distributed under the terms and Amyloid precursor protein (APP) transgenic mice show pathological changes resem- conditions of the Creative Commons bling those of AD patients [3–5]. Biochemical and histological studies have shown that Attribution (CC BY) license (https:// 2-month-old APPSw,Ind J9 transgenic mice do not exhibit Aβ in the hippocampus, which creativecommons.org/licenses/by/ begins to be detectable at 6 months of age, and by 12–18 months it is accumulated in senile 4.0/). plaques. Moreover, these transgenic mice show spatial memory deficits at 6 months in Int. J. Mol. Sci. 2022, 23, 13444. https://doi.org/10.3390/ijms232113444 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 13444 2 of 10 hippocampal-dependent memory tasks, such as Morris water maze (MWM) [6,7]. There- fore, the progression of biochemical, histological and behavioral changes over time make APPSw,Ind J9 mice an excellent murine model to compare early and late disease stages. Hippocampal dysfunction is one of the first alterations in early stages of AD, such as excitatory/inhibitory imbalance, synaptic plasticity disruption and memory deficits [2,8]. Given the proven neuronal hyperactivity in AD animal models [9], G-protein-gated in- wardly rectifying potassium (GIRK) channels have emerged as potential targets to regulate neuronal activity, based on their inhibitory role [10–12]. GIRK channels are a family of K+ channels activated by a variety of G-protein-coupled receptors, such as GABA, dopamine, serotonin or adenosine [12–14]. They form tetrameric units, assembled by a combination of their subunits GIRK1–GIRK4. In the brain, GIRK1, GIRK2 and GIRK3 are widely expressed throughout different areas, while GIRK4 subunit is classically related to the heart and its neuronal expression is limited [15]. Moreover, GIRK2 seems to be necessary for the correct function of these channels, and GIRK1/GIRK2 heteromers have been identified as the main GIRK channels in neurons [12,16]. Additionally, GIRK function and location depends on the effect of different modu- lators. Among those, the Regulator of G-protein signaling 7 (RSG7) is of special interest. It functions as a GTPase activating protein (GAP) that accelerates G-protein inactivation. Accordingly, mice lacking RGS7 show profoundly slow GIRK channel deactivation kinet- ics [17], disruption of inhibitory forms of synaptic plasticity and deficits in learning and memory [18]. Previous data from our group showed that in vitro acute incubation with Aβ solu- ble species induces depolarization of CA3 hippocampal pyramidal neurons by a loss-of- function of GIRK channels [19] and dysregulation of GIRK subunits genes (specifically, genes encoding GIRK2, GIRK3 and GIRK4) [20]. Furthermore, increasing GIRK activity restores hippocampal Long-Term Potentiation (LTP) and memory deficits induces by Aβ in an in vivo mouse model of amyloidosis [21–23]. However, all this promising data have been obtained in an intracerebroventricular murine model of early amyloidosis, when Aβ is soluble rather than accumulated. A recent study has provided evidence of GIRK redistribution from the plasma membrane to intracellular sites and pre- and post-synaptic reduction in GIRK2 channels in two transgenic AD models, P301S and APP/PS1 [24]. Nevertheless, it is unknown whether GIRK subunits levels are early altered in the brain of mice developing AD pathological changes that include progressive age dependent Aβ accumulation. Furthermore, it is well-established that periodic cognitive training on a hippocampal-dependent memory task may mitigate memory deficits observed in AD, both in rodents [7,25,26] and humans [27,28], as well as attenuate Aβ deposition and enhance adult hippocampal neurogenesis [29]. Accordingly, training in a spatial memory task might be able to reverse early alterations in GIRK expression caused by Aβ-overexpression, with the subsequent recovery of hippocampal function. Thus, the aim of the present study was to determine the effect of Aβ accumulation (due to genotype and age) and training on the protein expression of GIRK subunits and its modulator, in the hippocampus of APPSw,Ind J9 transgenic mice. 2. Results The experimental design of this study is detailed in Figure 1. Firstly, we examined the protein levels of GIRK channel subunits and RGS7 in the hippocampus of adult APPSw,Ind J9 transgenic mice at early (6 months) and late (12–18 months) pathological stages that differ in hippocampal Aβ accumulation levels [7]. As shown in Figure 2A, APPSw,Ind J9 mice showed intracellular Aβ accumulation in the hippocampus at 6 months and amyloid plaques at 12–18 months. Thus, 6 months of age in this murine model is an adequate choice to study early disease stages vs. late ones (12–18 months). In addition, APPSw,Ind J9 mice showed a memory impairment at 6 months of age, as data from the MWM showed a higher latency to find the platform (Figure 2B; F(1,6) = 21.23; p = 0.0037) in the transgenic group Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 3 of 11 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 3 of 11 Int. J. Mol. Sci. 2022, 23, 13444 3 of 10 APPSw,Ind J9 mice showed a memory impairment at 6 months of age, as data from the APP MWM Sw,Ind J9 mice showed a memory impairment at 6 months of age, as data from the showed a higher latency to find the platform (Figure 2B; F(1,6) = 21.23; p = 0.0037) in MWM compared showed acontrol, higher to thegroup the transgenic latency as wellto compared astofind athe the lower platform number control, (Figure as 2B; of crossings as well F(1,6) =number 21.23; p to specifically a lower = 0.0037) of the in target crossings the transgenic quadrant (TQ; specifically group to Figure compared 2C;quadrant the target to the control, p = 0.0154). t(3) = 5.00;(TQ; as well as a lower number Figure 2C; t(3) = 5.00; p = 0.0154). of crossings specifically to the target quadrant (TQ; Figure 2C; t(3) = 5.00; p = 0.0154). Figure1.1.Experimental Figure Experimental design design showing showing number number of of animals animals for for each each condition, condition, representation representation ofof Figure training1.inExperimental design the Morris Water showing Maze number (MWM), of animals hippocampal for each dissection condition, for Westernrepresentation of blot analysis, and training trainingin inthe theMorris Morris Water Water Maze Maze (MWM), (MWM), hippocampal dissectionfor hippocampal dissection forWestern Westernblot blotanalysis, analysis,and and corresponding figure numbers. corresponding correspondingfigure figurenumbers. numbers. Figure 2. Figure 2. Age-dependent Age-dependent progression progression of of amyloid amyloid pathology. pathology. (A) (A) Age-dependent Age-dependent amyloid amyloid Figure 2. Age-dependent accumulation in the progression the hippocampus hippocampus of amyloid of APP APP pathology. (A) Age-dependent amyloid accumu- Sw,Ind J9 mice at 6, 12 and 18 months of age. Brain sections accumulation in of Sw,Ind J9 mice at 6, 12 and 18 months of age. Brain sections lation in the hippocampus were stained stained with an an anti-Aβ anti-Aβ of APP 6E10Sw,Ind J9 mice antibody. at bar: Scale 6, 12250 andµm. 18 months m,months; months;of age. BrainWT, sections were were with 6E10 antibody. Scale bar: 250 µm. m, h,h,hours; hours; WT, wild wildtype. type. stained with to anfindanti-Aβ 6E10 antibody. Scale bar: 250 µm. 5m, months; h, hours; WT, wild type. (B) Latency to find the hidden platform in the MWM during 5 days of training. Data is expressed asas (B) Latency the hidden platform in the MWM during days of training. Data is expressed mean (B) ±± SEM. Latency mean SEM. s, seconds. to s, findseconds. the hidden ** pp <