https://orcid.org/0000-0003-0712-0788
Abstract
Combination treatments based on pharmacological interactions at α7 nicotinic acetylcholine receptors (nAChRs) are promising therapeutic approaches for neurocognitive disorders.
Here, we tested the cognitive efficacy of combinations of memantine with an α7 nAChR-selective agonist (PHA-543613) in naturally aged rats. Age-related changes in the expression of some key genes and proteins were also measured using quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).
Aged rats showed marked cognitive decline in the novel object recognition test, and they also exhibited cholinergic changes such as mRNA upregulation of α7 nAChRs. Upregulation of interleukin-1β, macrophage inflammatory protein 1α, CX3CL1, intercellular adhesion molecule 1, and ciliary neurotrophic factor mRNA was also detected in aged rats. Combination treatment of memantine and PHA-543613 successfully alleviated the age-related decline of recognition memory of rats by exceeding the effects of the corresponding monotreatments.
Results indicate a positive interaction between memantine and PHA-543613, which also reflects a putative role of α7 nAChRs in the cognitive enhancer effects of memantine. These findings may facilitate the development of combination therapies for age-related neurocognitive disorders.
Neurocognitive disorders (NCDs) such as Alzheimer’s disease (AD) are widely prevalent among the elderly population; however, currently approved pharmacological treatments provide only limited effectiveness. Recent studies suggested that therapeutic efficacy may be improved by applying combination therapies. Here, we tested the combination of multi-target AD medication memantine and the alpha7 nicotinic acetylcholine receptor (nAChR) agonist PHA-543613 in aged rats. Aged rats were impaired in the novel object recognition declarative memory task, coupled with higher mRNA expression of various neuroinflammatory markers and the alpha7 nAChR genes. Memory performance was improved by a low-dose combination of memantine and PHA-543613 producing an effectiveness superior to corresponding monotreatments. These results support the notion that combination therapies may open new avenues in the treatment of NCDs. Furthermore, the present results also strongly suggest that the previously suggested high affinity of memantine to alpha7 nAChR may also be important in its mechanism of action.
INTRODUCTION
Aging of the brain is generally associated with progressive neurocognitive disorders (NCDs), that pose serious public health challenges worldwide.1,2 Currently, multi-target, glutamatergic N-methyl-D-aspartate (NMDA) receptor antagonist memantine and different cholinesterase inhibitors (eg, donepezil) are the only approved therapeutic options for patients with NCDs; however, several clinical observations indicate that these medications may provide only moderate and transient symptomatic benefits with very limited or no disease-modifying potential.3-5 Therefore, the development of new treatment strategies is still an unmet medical need, and the discovery of new treatments would be crucial in the field.6 The alpha7 nicotinic acetylcholine receptor (α7 nAChR) is considered a promising target to treat NCDs because it is primarily involved in learning and memory processes7,8 and also plays a role in immune regulation.9 It is well known that signaling through α7 nAChRs enhances both cholinergic and glutamatergic transmission contributing to memory improvement in preclinical disease models.10-12 In addition, in recent years, stimulation of α7 nAChRs expressed by glial cells has been shown to reduce neuroinflammation by activating the cholinergic anti-inflammatory pathway.8,13-16
Combination therapies may be highly promising novel approaches in the treatment of age-related NCDs, since the co-application of pharmacological agents with different mechanisms of action may result in a synergistic increase in efficacy and/or enable the use of lower doses while achieving the same effectiveness with less side-effects. Furthermore, combination therapies may offer a complex influence on the dysfunction of signaling pathways and other pathological processes involved in NCDs,17 which may add up in beneficial disease-modifying pharmacological effects. Available preclinical results suggest promising therapeutic outcome in cases when memantine is combined with different compounds acting directly on the α7 nAChRs18 compared to less effective combinations of memantine and acetylcholine esterase inhibitor (AChEI) donepezil.19,20 For example, co-administration of memantine with galantamine—which acts not only as an AChEI but also as a positive allosteric modulator (PAM) of α7 nAChRs—alleviates scopolamine-induced memory deficits in mice21 and delays natural forgetting in rats22 with higher efficacy compared to the corresponding monotreatments. These findings are further supported by 3 recent studies from our laboratory, in which the combined effects of memantine and the selective α7 nAChR agonist PHA-543613 were tested on different cognitive functions in a scopolamine-induced transient amnesia model. First, we reported that rats under the combined memantine-PHA-543613 treatment showed better working memory performance in the spontaneous alternation task compared to monotreatments.23 Second, our recent findings also indicate that the same treatment combination (ie, memantine-PHA-543613) successfully improved both short-term memory and recall of long-term memory of rats in the Morris water maze test.24 Third, our recent in vivo electrophysiological experiments demonstrated that the memantine-PHA-543613 combination increased the ACh-evoked firing activity of CA1 hippocampal pyramidal cells.25 Based on the above findings, we previously suggested a potential pharmacodynamic interaction between α7 nAChR activator ligands and memantine, which was also supported by previous studies that reported higher affinity of memantine to α7 nAChRs than to NMDA receptors.26,27
Taken together, the aim of the current study was to further investigate the behavioral level interaction between memantine and a selective α7 nAChR agonist PHA-543613 in a preclinical animal model of age-related cognitive deficit. In the present study, naturally aged rats were used as they show age-related cognitive impairment and certain emerging pathological processes including neuroinflammation and apoptotic cell death, posing as one of the most relevant models of spontaneous, age-related cognitive decline in humans.28,29 Since aging is not a uniform process neither in rodents nor in humans, our further aim was to assess key molecular-level indicators of pathological and healthy aging animals by investigating the mRNA and protein expression of inflammatory and other immunological biomarkers, neurotrophic factors, and α7 nAChR in both memory impaired and unimpaired animals.
MATERIALS AND METHODS
Animals
Altogether 39 (comprising of 11-13 subjects/group) naturally aged (28 m. o.) and 12 young (4 m.o.) male Long Evans rats (Charles River Laboratories) were applied in the current study. Animals were pair-housed under a 12/12-h daily light/dark cycle (lights ON from 7 AM to 7 PM) with controlled temperature and humidity in the animal house of the Szentágothai Research Centre, University of Pécs, Hungary. The animals were tested in the light period and were fed daily after the experiments with a controlled amount (17 g/animal/day) of dry laboratory rodent chow (ssniff-Spezialdiäten GmbH) to prevent the development of obesity and other related health problems. Water was available ad libitum. All experiments were approved by the Animal Welfare Committee of the University of Pécs, and the National Scientific Ethical Committee on Animal Experimentation (ÁTET) at the Ministry of Agriculture (license no.: BA02/2000–25/2015, BA02/2000-30/2021, Issued by the Baranya County Office of the Hungarian Government). All procedures fully complied with the Decree No. 40/2013 (II. 14.) of the Hungarian Government and the EU Directive 2010/63/EU on the Protection of animals used for scientific purposes.
Behavioral Assessment
Novel Object Recognition Test
Long-term declarative memory of the animals was evaluated with the novel object recognition (NOR) test paradigm. The experiments were carried out as described in previous reports from our laboratory.30,31 In brief, a standard open field box was used made of gray-colored plywood, in size of 57.5 × 57.5 cm (length × width) surrounded by 39.5 cm high walls. The test protocol consisted of 2 trials: an acquisition trial followed by a test trial after 24-h retention interval. In the acquisition trial, 2 identical objects were placed near the left and right corners of the open field box, and the rats were allowed to explore the environment and the objects for 3 min. After the 24-h retention interval (spent in the home cage), in the test trial, object exploration was assessed again for 3 min, where one of the objects was changed to a novel one. Four different object pairs were used that were distributed randomly between animals and experimental sessions in a counterbalanced, Latin-square, within-subject experimental design.
The time spent with the familiar (Ef) and the novel (En) objects was recorded, and a discrimination index (DI) was calculated based on the following formula32:
Rats who did not observe the 2 objects together for at least 5 s or observed only one of the objects for the entire duration of the test trial (ie, DI=±1.00) were excluded from the analysis of the given session.
Drugs and Routes of Administration
PHA-543613 hydrochloride (Cat. No. 3092, Tocris) and memantine hydrochloride (Cat. No. 0773, Tocris) were dissolved in physiological saline (0.9% NaCl solution) and were applied in a final injection volume of 1 mL/kg. All compounds were injected subcutaneously at 45 min before the acquisition trial. In co-administration treatments, memantine and PHA-543613 were administered consecutively in 2 separate subcutaneous injections. When monotreatments were tested, drugs that were not administered were replaced with the corresponding vehicle.
Experimental Design
First, the baseline performance of aged and young rats was compared. After the successful demonstration of cognitive impairment in aged rats, the pharmacological effects of monotreatments with memantine and PHA-543613, as well as their co-administration were tested in aged animals. In the pharmacological experiments, treatments were applied in a counterbalanced, within-subject design to achieve a fully randomized sequence of different treatments. Monotreatments with memantine were applied in the following doses: 0.1 mg/kg, 0.3 mg/kg, and 1.0 mg/kg (Mem0.1, Mem0.3, and Mem1.0, respectively). PHA-543613 was administered in doses of 0.3 mg/kg, 1.0 mg/kg, and 3.0 mg/kg (PHA0.3, PHA1.0, and PHA3.0, respectively). Then, co-administration of subeffective memantine and PHA-543613 doses was tested against the effects of the corresponding monotreatments by applying the following treatments in a counterbalanced Latin-square design: saline vehicle alone (VEH), memantine monotreatment in 0.01 mg/kg dose (Mem0.01), PHA-543613 monotreatment in 0.1 mg/kg dose (PHA0.1), and co-administration treatment (Mem0.01&PHA0.1). To ensure that no residual effects of the compounds influenced the results, we included a minimum 2-day interval between experimental sessions, allowing for complete washout (in rats, memantine has a half-life of < 4 h, PHA-543613 has a half-life of 40 min).33,34
Brain Tissue Collection and Preparation
Aged animals were divided into memory impaired (AI) and unimpaired (AU) aged groups depending on their baseline cognitive performance. The grouping of AU and AI rats was based on the median baseline performance (DI) of all aged rats (N = 39) in the NOR test after vehicle treatment, which was DI = 0.087. Rats who performed above the median DI were considered AU, while rats with a DI lower than the median were considered AI rats. Five AU rats (DI range: 0.17-0.68) and 6 AI rats (DI range: –0.66 to –0.01) were chosen for biochemical analysis from experiments that investigated different treatments. At least 6 days elapsed between the last drug administration session and the perfusion of the animals. For post mortem analysis, animals were anesthetized with an overdose of pentobarbital and were transcardially perfused with saline, and their brains were rapidly removed and dissected into left and right neocortex (CTX), striatum (STR), and hippocampus (HC). The brain samples were frozen immediately in liquid nitrogen and were stored at −80 oC until biochemical analyses were performed. During the preparation of RNA and protein samples, whole subcortical and archicortical brain regions (STR and HC) or random samples of the neocortex (CTX) were used.
RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR
The total RNA of the brain samples was extracted according to the manufacturer’s protocol by applying the NucleoSpin RNA kit (MACHEREY-NAGEL). The quality and quantity of RNA were assessed at 260 nm using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). The cDNA was constructed from total RNA with High Capacity cDNA reverse transcription kit (Thermo Fisher Scientific) in 20 μL reactions using random hexamers following the manufacturer’s protocol. The resulting cDNA was stored at −20 °C. Target gene expressions were measured using real-time PCR using Maxima SYBRGreen MasterMix (Applied Biosystems) with an ABI Prism 7500 instrument (Applied Biosystems). The cDNAs were applied as a template for the amplification reactions. All samples were tested in duplicates. Primers were designed by Primer Express software (Thermo Fisher Scientific) considering the exon-intron boundaries for all target genes (Table 1). Cyclophilin A was used as a housekeeping gene for the quantification of RNA. The thermal profile started at 95 °C for 10 min, 40 cycles of 35 s at 95 °C, 35 s at 60 °C, and 1 min at 72 °C.
Primer sequences applied for qPCR.
| Target gene | GenBank Acc# | Primers (5′-3′)a | Amplicon size (bp) |
|---|---|---|---|
| Cyclophilin A | BC059141 | GGA AGC CAT GGA GCG TTT T AAT GCC CGC AAG TCA AAG AA | 100 |
| IL-1β | NM_031512 | GAG TCT GCA CAG TTC CCC AA ATG TCC CGA CCA TTG CTG TT | 100 |
| MIP-1α | NM_013025 | AGC CGG GTG TCA TTT TCC T CCA GCT CAG TGA TGT ATT CTT GGA | 90 |
| CX3CL1 | NM_134455 | TGG CCG CGT TCT TTC ATC GAT TGG CGA GGT CAT CTT GTG | 95 |
| ICAM-1 | NM_012967 | GAA GAC AGC AGA CCA CTG TGC TT TCG CTC TGG GAA CGA ATA CAC | 90 |
| CNTF | X17457 | AGG CCC CGA GAG CAA AAT ACC ACC CAA TGC AAA CCA AA | 90 |
| BDNF | M61175 | CAC TTT TGA GCA CGT GAT CGA CAC CCG GGA AGT GTA CAA GTC | 100 |
| Nicotinic receptor α7 | S53987 | AGT GCT GCA AAG AGC CAT ACC ATG AGT ACA CAA GGG ATG AGC AGA T | 100 |
| Target gene | GenBank Acc# | Primers (5′-3′)a | Amplicon size (bp) |
|---|---|---|---|
| Cyclophilin A | BC059141 | GGA AGC CAT GGA GCG TTT T AAT GCC CGC AAG TCA AAG AA | 100 |
| IL-1β | NM_031512 | GAG TCT GCA CAG TTC CCC AA ATG TCC CGA CCA TTG CTG TT | 100 |
| MIP-1α | NM_013025 | AGC CGG GTG TCA TTT TCC T CCA GCT CAG TGA TGT ATT CTT GGA | 90 |
| CX3CL1 | NM_134455 | TGG CCG CGT TCT TTC ATC GAT TGG CGA GGT CAT CTT GTG | 95 |
| ICAM-1 | NM_012967 | GAA GAC AGC AGA CCA CTG TGC TT TCG CTC TGG GAA CGA ATA CAC | 90 |
| CNTF | X17457 | AGG CCC CGA GAG CAA AAT ACC ACC CAA TGC AAA CCA AA | 90 |
| BDNF | M61175 | CAC TTT TGA GCA CGT GAT CGA CAC CCG GGA AGT GTA CAA GTC | 100 |
| Nicotinic receptor α7 | S53987 | AGT GCT GCA AAG AGC CAT ACC ATG AGT ACA CAA GGG ATG AGC AGA T | 100 |
aUpper and lower sequences represent forward and reverse primers, respectively.
Abbreviations: BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; ICAM-1, intercellular adhesion molecule 1; IL-1β, interleukin-1β.
Primer sequences applied for qPCR.
| Target gene | GenBank Acc# | Primers (5′-3′)a | Amplicon size (bp) |
|---|---|---|---|
| Cyclophilin A | BC059141 | GGA AGC CAT GGA GCG TTT T AAT GCC CGC AAG TCA AAG AA | 100 |
| IL-1β | NM_031512 | GAG TCT GCA CAG TTC CCC AA ATG TCC CGA CCA TTG CTG TT | 100 |
| MIP-1α | NM_013025 | AGC CGG GTG TCA TTT TCC T CCA GCT CAG TGA TGT ATT CTT GGA | 90 |
| CX3CL1 | NM_134455 | TGG CCG CGT TCT TTC ATC GAT TGG CGA GGT CAT CTT GTG | 95 |
| ICAM-1 | NM_012967 | GAA GAC AGC AGA CCA CTG TGC TT TCG CTC TGG GAA CGA ATA CAC | 90 |
| CNTF | X17457 | AGG CCC CGA GAG CAA AAT ACC ACC CAA TGC AAA CCA AA | 90 |
| BDNF | M61175 | CAC TTT TGA GCA CGT GAT CGA CAC CCG GGA AGT GTA CAA GTC | 100 |
| Nicotinic receptor α7 | S53987 | AGT GCT GCA AAG AGC CAT ACC ATG AGT ACA CAA GGG ATG AGC AGA T | 100 |
| Target gene | GenBank Acc# | Primers (5′-3′)a | Amplicon size (bp) |
|---|---|---|---|
| Cyclophilin A | BC059141 | GGA AGC CAT GGA GCG TTT T AAT GCC CGC AAG TCA AAG AA | 100 |
| IL-1β | NM_031512 | GAG TCT GCA CAG TTC CCC AA ATG TCC CGA CCA TTG CTG TT | 100 |
| MIP-1α | NM_013025 | AGC CGG GTG TCA TTT TCC T CCA GCT CAG TGA TGT ATT CTT GGA | 90 |
| CX3CL1 | NM_134455 | TGG CCG CGT TCT TTC ATC GAT TGG CGA GGT CAT CTT GTG | 95 |
| ICAM-1 | NM_012967 | GAA GAC AGC AGA CCA CTG TGC TT TCG CTC TGG GAA CGA ATA CAC | 90 |
| CNTF | X17457 | AGG CCC CGA GAG CAA AAT ACC ACC CAA TGC AAA CCA AA | 90 |
| BDNF | M61175 | CAC TTT TGA GCA CGT GAT CGA CAC CCG GGA AGT GTA CAA GTC | 100 |
| Nicotinic receptor α7 | S53987 | AGT GCT GCA AAG AGC CAT ACC ATG AGT ACA CAA GGG ATG AGC AGA T | 100 |
aUpper and lower sequences represent forward and reverse primers, respectively.
Abbreviations: BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; ICAM-1, intercellular adhesion molecule 1; IL-1β, interleukin-1β.
Protein Isolation and ELISA Test
Brain homogenates were prepared in RIPA lysis buffer (50 mM Tris–HCl, pH: 8.0, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 5 mM EDTA, 0.1% SDS). The total protein concentrations of the samples were measured using a BCA Protein Assay kit (Pierce). Quantification of interleukin-1β (IL-1β), macrophage inflammatory protein 1α (MIP-1α), intercellular adhesion molecule 1 (ICAM-1), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and α7 nAChR was carried out using Abbexa ELISA kits with the following catalog numbers, respectively: abx155713, abx155822, abx155662, abx155251, abx155360, abx556026. Quantification of fractalkine (CX3CL1) was carried out using Abcam ELISA kit (Cat. No. ab100761). ELISAs were performed according to the manufacturers’ protocols. Immediately after developing the final color reaction of the assay, the absorbance was measured at 450 nm with an iEMS Reader microplate reader (Thermo Fisher Scientific).
Statistical Analysis
Data were expressed as mean ± SEM. Statistical analyses were performed using the IBM SPSS 20.0 software (IBM). In the NOR test, the time spent exploring the familiar and novel objects was compared using paired samples T-test to assess whether rats after a given treatment show normal recognition memory performance. The DIs of aged and young animals were compared using independent samples T-test. In pharmacological experiments, the main effects of treatments on DI were analyzed using a linear mixed-effects model. Following a significant main effect, the effects of the tested treatments were compared with the effects of the vehicle using an uncorrected post hoc test. RNA and protein expression levels of young, memory impaired, and unimpaired aged groups were compared using univariate analysis of variance (ANOVA) and post hoc least significant difference (LSD) test.
RESULTS
Effects of Aging on Long-Term Recognition Memory Performance
To determine the age-related cognitive impairment of rats, novel object recognition memory of the aged and young experimental groups was assessed and compared. In the test trial after 24-h retention interval, young animals spent significantly more time exploring the novel object compared to the familiar one (n = 12; exploration time, novel vs familiar: 9.8 ± 0.9 s vs 4.6 ± 0.4 s, t = 6.666, df = 11, P < .001). As expected, no such discrimination between the familiar and the novel objects was observed in the aged rats (n = 11; novel vs familiar: 8.1 ± 1.0 s vs 6.1 ± 0.7 s, t = 1.526, df = 10, P = .158) (Figure 1A). In addition, the DI of aged rats was significantly lower compared to young rats (naged = 11, nyoung = 12; aged vs young: 0.130 ± 0.085 vs 0.352 ± 0.039, t = 2.426, df = 21, P = .024) (Figure 1B). Results indicate that aged rats show remarkable deficits in their long-term declarative (recognition) memory performance.
Comparison of the performance of aged and young animals in the NOR test (naged = 11, nyoung = 12). (A) Young rats spent significantly more time exploring the novel object compared to the familiar one, while aged rats did not discriminate between the novel and the familiar objects. (B) Discrimination index (DI) of aged rats was significantly lower compared to young rats. Asterisks mark significant differences in the time spent with the exploration of the novel and the familiar objects: ***P < .001 (paired samples T-test). Hash indicates a significant difference between the DI of aged and young animals: #P < .05 (independent samples T-test). NOR, novel object recognition.
Cognitive Enhancer Effects of Memantine and PHA-543613 Monotreatments
Unlike the vehicle treatment, the administration of memantine (0.1 and 1.0 mg/kg) resulted in the significantly longer exploration of the novel object as compared to the familiar object (n = 12; exploration time, novel vs familiar: VEH: 9.3 ± 1.6 s vs 7.3 ± 1.1 s, t = 1.489, df = 10, P = .167; Mem0.1: 9.0 ± 1.3 s vs 5.5 ± 0.5 s, t = 2.492, df = 11, P = .030; Mem1.0: 8.7 ± 1.2 s vs 5.7 ± 1.0 s, t = 2.737, df = 10, P = .021). Thus, 0.1 and 1.0 mg/kg doses of memantine reversed the age-related recognition memory deficit in rats. However, the effect of the memantine monotreatments on the DI was not significant (n = 12; main effect of treatment: F(3, 32) = 0.384, P = .765) (Figure 2B).
Effects of monotreatments with memantine and PHA-543613 on the performance of aged rats in the NOR test. (A) Aged rats (n = 12) that received memantine in the lowest (0.1 mg/kg) and in the highest (1.0 mg/kg) doses showed preference toward the exploration of the novel object compared to the familiar one. (B) DI was not significantly increased by memantine treatment. (C) Aged rats (n = 13) that received PHA-543613 in 0.3 mg/kg and 1.0 mg/kg doses explored the novel object for a significantly longer time compared to the familiar one. (D) PHA-543613 in the lowest dose (0.3 mg/kg) improved DI of aged rats compared to the corresponding vehicle treatment. Asterisks mark significant differences in the time spent with the exploration of the novel and the familiar objects: ***P < .001, *P < .05 (paired samples T-test). Hash indicates significantly different DI compared to the corresponding vehicle treatment: #P < .05 (linear mixed-effect model + post hoc LSD). DI, discrimination index; NOR, novel object recognition.
Aged animals receiving PHA-543613 in 0.3 mg/kg or 1.0 mg/kg doses showed a preference toward the novel object (n = 13; novel vs familiar: PHA0.3: 10.4 ± 0.8 s vs 5.0 ± 0.8 s, t = 6.354, df = 12, P < .001; PHA1.0: 9.3 ± 1.2 s vs 6.7 ± 0.8 s, t = 2.684, df = 12, P = .020), while aged rats when treated with vehicle did not discriminate between the objects (novel vs familiar: VEH: 9.4 ± 1.7 s vs 7.8 ± 1.1 s, t = 0.799, df = 10, P = .443) (Figure 2C). Furthermore, PHA-543613 at the lowest dose (0.3 mg/kg) improved the DI of the aged animals compared to the vehicle treatment (n = 13; main effect of treatment: F(3, 34.7) = 4.189, P = .012; PHA0.3 vs VEH: 0.38 ± 0.07 vs 0.05 ± 0.12, P = .012) (Figure 2D).
Co-Administration of Memantine With PHA-543613
Results showed that aged rats after the treatment with memantine alone and in combination with PHA-543613 spent more time exploring the novel object than the familiar object (n = 12; novel vs familiar: Mem0.01: 8.1 ± 0.8 s vs 5.4 ± 0.5 s, t = 2.549, df = 10, P = .029; Mem0.01&PHA01: 10.1 ± 1.2 s vs 5.0 ± 0.6 s, t = 3.544, df = 11, P = .005) in contrast to the vehicle treatment (novel vs familiar: VEH: 6.5 ± 1.0 s vs 7.2 ± 1.1 s, t=−0.465, df = 10, P = .652), and the monotreatment with PHA-543613 which were not found effective (novel vs familiar: PHA0.1: 8.2 ± 1.3 s vs 6.7 ± 1.1 s, t = 1.604, df = 10, P = .140) (Figure 3A). The applied treatments resulted in a marginal effect on DI (n = 12; main effect of treatment: F(3, 30) = 2.727, P = .062). As expected, post hoc pairwise comparisons did not show any significant increase of DI by the low-dose monotreatments with memantine or PHA-543613 compared to the vehicle treatment (Mem0.01 vs VEH: 0.19 ± 0.07 vs − 0.06 ± 0.12, P = .081; PHA01 vs VEH: 0.09 ± 0.07 vs − 0.06 ± 0.12, P = .298). In contrast, DI was significantly increased by the combination treatment (Mem0.01&PHA01 vs VEH: 0.31 ± 0.10 vs − 0.06 ± 0.12, P = .01) indicating that the combination of the 2 putatively distinct cellular mechanisms of action passed a threshold to successfully reverse recognition memory decline of aged rats (Figure 3B).
Effects of combination treatments with memantine and PHA-543613 on the performance of aged rats in the NOR test (n = 12). (A) Aged rats that received memantine alone and in combination with PHA-543613 spent more time exploring the novel object compared to the familiar one. (B) The memantine-PHA-543613 combination treatment significantly improved the DI of aged rats compared to the vehicle treatment. Asterisks mark significant differences between the exploration time of the novel and the familiar objects: **P < .01, *P < .05 (paired samples T-test). Hash indicates significantly different DI compared to the corresponding vehicle treatment: #P < .05 (linear mixed-effect model + post hoc LSD). DI, discrimination index; NOR, novel object recognition.
Effects of Aging on Brain mRNA Expression Levels of Selected Genes
Results of quantitative real-time PCR (RT-PCR) analysis revealed that both AU and AI animals showed significantly higher IL-1β mRNA levels in the neocortex and the STR compared to young control rats (CTX: F(2, 11) = 5.631, P = .021; AI (n = 5) vs young (n = 5): 26.9 ± 4.7 vs 9.7 ± 2.1, P = .008; AU (n = 4) vs young: 22.2 ± 4.5 vs 9.7 ± 2.1, P = .047; STR: F(2, 11) = 8.753, P = .005, AI (n = 5) vs young (n = 5): 55.3 ± 8.3 vs 18.9 ± 2.4, P = .002; AU (n = 4) vs young: 40.8 ± 7.2 vs 18.9 ± 2.4, P = .038) (Figure 4A and B). On the contrary, only AI animals exhibited a significant increase of IL-1β mRNA in the HC compared to young animals (n = 5/group; F(2, 12) = 6.708, P = .013; AI vs young: 35.6 ± 3.1 vs 19.1 ± 3.6, P = .005), and a difference between AU and young groups was not found (AU vs Young: 23.7 ± 3.4 vs 19.1 ± 3.6, P = .349) (Figure 4C).
Brain mRNA expression levels of inflammatory markers, neurotrophic factors, and the α7 nAChR in young, and cognitively unimpaired (AU) or impaired (AI) aged rats. (A-C) Neocortical (nyoung = 5, nAU = 4, nAI = 5), striatal (nyoung = 5, nAU = 4, nAI = 5), and hippocampal (nyoung = 5, nAU = 5, nAI = 5) IL-1β mRNA levels. (D-F) Neocortical (nyoung = 5, nAU = 5, nAI = 5), striatal (nyoung = 5, nAU = 5, nAI = 5), and hippocampal (nyoung = 5, nAU = 5, nAI = 5) MIP-1α mRNA levels. (G-I) Neocortical (nyoung = 5, nAU = 5, nAI = 5), striatal (nyoung = 5, nAU = 4, nAI = 5), and hippocampal (nyoung = 5, nAU = 5, nAI = 5) CX3CL1 mRNA levels. (J-L) Neocortical (nyoung = 5, nAU = 4, nAI = 5), striatal (nyoung = 5, nAU = 4, nAI = 5), and hippocampal (nyoung = 5, nAU = 5, nAI = 5) ICAM-1 mRNA levels. (M-O) Neocortical (nyoung = 5, nAU = 4, nAI = 5), striatal (nyoung = 5, nAU = 5, nAI = 5), and hippocampal (nyoung = 5, nAU = 5, nAI = 5) CNTF mRNA levels. (P-R) Neocortical (nyoung = 5, nAU = 5, nAI = 5), striatal (nyoung = 5, nAU = 5, nAI = 5), and hippocampal (nyoung = 5, nAU = 5, nAI = 5) BDNF mRNA levels. (S-U) Neocortical (nyoung = 5, nAU = 4, nAI = 5), striatal (nyoung = 5, nAU = 4, nAI = 5), and hippocampal (nyoung = 5, nAU = 5, nAI = 5) α7 nAChR mRNA levels. Asterisks mark significant changes in mRNA expression levels compared to young animals: ***P < .001, **P < .01, *P < .05 (univariate ANOVA + post hoc LSD). CHRNA7, α7 nicotinic acetylcholine receptors; BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; ICAM-1, intercellular adhesion molecule 1; IL-1β, interleukin-1β; MIP-1α, macrophage inflammatory protein 1α.
Macrophage inflammatory protein 1α mRNA expression was upregulated in both AU and AI rats in all selected brain areas compared to young animals (n = 5/group in each brain area; CTX: F(2, 12) = 5.860, P = .017; AI vs young: 75.6 ± 20.7 vs 14.5 ± 3.8, P = .006; AU vs young: 58.3 ± 8.0 vs 14.5 ± 3.8, P = .035; STR: F(2, 12) = 45.5, P < .001; AI vs young: 184.8 ± 22.7 vs 42.6 ± 5.8, P < .001; AU vs young: 232.1 ± 9.7 vs 42.6 ± 5.8, P < .001; HC: F(2, 12) = 16.121, P < .001; AI vs young: 122.1 ± 21.1 vs 18.8 ± 1.6, P > 0.001; AU vs young: 96.2 ± 9.5 vs 18.8 ± 1.6, P = .001) (Figure 4D-F). In the neocortex and the HC, the AI group showed the highest mean expression of MIP-1α mRNA (Figure 4D and F).
The upregulation of mRNA expression of the other selected chemokine CX3CL1 was observed only in the AI rats compared to young rats in all examined brain regions (CTX: F(2, 12) = 5.420, P = .021; AI (n = 5) vs young (n = 5): 32.7 ± 3.1 vs 24.9 ± 0.6, P = .037; STR: F(2, 11) = 11.824, P = .002; AI (n = 5) vs young (n = 5): 53.6 ± 5.2 vs 29.3 ± 3.1, P = .003; HC: F(2, 12) = 10.665, P = .002; AI (n = 5) vs young (n = 5): 31.3 ± 2.9 vs 18.7 ± 1.1, P = .001) (Figure 4G-I), while CX3CL1 mRNA level was similar in AU and young animals (CTX: AU (n = 5) vs young: 22.1 ± 2.5 vs 24.9 ± 0.6, P = .423; STR: AU (n = 4) vs young: 24.2 ± 5.4 vs 29.3 ± 3.1, P = .467; HC: AU (n = 5) vs young: 21.1 ± 1.8 vs 18.7 ± 1.1, P = .435).
The striatal and neocortical level of ICAM-1 mRNA was higher in both AI and AU groups than in the young group (CTX: F(2, 11) = 10.636, P = .003; AI (n = 5) vs young (n = 5): 46.7 ± 6.6 vs 16.2 ± 1.8, P = .001; AU (n = 4) vs young: 33.1 ± 4.8 vs 16.2 ± 1.8, P = .035; STR: F(2, 11) = 9.561, P = .004; AI (n = 5) vs young (n = 5): 41.4 ± 4.4 vs 20.8 ± 2.7, P = .001; AU (n = 4) vs young: 35.6 ± 3.1 vs 20.8 ± 2.7, P = .016) (Figure 4J and K). In the HC, only the AI rats showed elevated ICAM-1 mRNA expression level compared to the young rats (n = 5/group; HC: F(2, 12) = 11.4, P = .002; AI vs young: 61.7 ± 6.3 vs 35.8 ± 2.2, P = .001) (Figure 4L). Hippocampal ICAM-1 mRNA level was not different between AU and young animals (HC: AU vs young: 40.7 ± 2.4 vs 35.8 ± 2.2, P = .411).
Ciliary neurotrophic factor mRNA expression levels were significantly increased in the AI group in the neocortex and HC compared to young rats (CTX: F(2, 11) = 4.167, P = .045; AI (n = 5) vs young (n = 5): 12.2 ± 2.3 vs 6.4 ± 0.4, P = .017; HC: F(2, 12) = 4.099, P = .044; AI (n = 5) vs young (n = 5): 46.0 ± 8.2 vs 28.1 ± 1.7, P = .029). In contrast, AU rats did not express increased mRNA levels of CNTF in these brain regions (CTX: AU (n = 4) vs young: 7.9 ± 0.8 vs 6.4 ± 0.4, P = .507; HC: AU (n = 5) vs young: 28.0 ± 2.8 vs 28.1 ± 1.7, P = .990) (Figure 4M and O). Ciliary neurotrophic factor mRNA expression in the STR did not differ between the groups (n = 5/group; F(2, 12) = 0.394, P = .683) (Figure 4N). Furthermore, we did not find any significant difference in the expression levels of BDNF mRNA between the groups in any of the tested brain areas (n = 5/group; CTX: F(2, 12) = 0.081, P = .922; STR: F(2, 9) = 1.786, P = .222; HC: F(2, 12) = 1.057, P = .378) (Figure 4P-R).
Memory impaired rats also showed a tendency to upregulated α7 nAChR mRNA levels in the examined brain areas compared to the young group, while the AU group expressed α7 nAChR mRNA levels similar to the young group (CTX: F(2, 11) = 3.739, P = .058; AI (n = 5) vs young (n = 5): 28.6 ± 4.6 vs 15.8 ± 1.3, P = .020; AU (n = 4) vs young: 20.7 ± 3.2 vs 15.8 ± 1.3, P = .350; STR: F(2, 11) = 5.635, P = .021; AI (n = 5) vs young (n = 5): 16.1 ± 0.9 vs 13.5 ± 0.7, P = .027; AU (n = 4) vs young: 12.7 ± 0.4 vs 13.5 ± 0.7, P = .489; HC: F(2, 12) = 3.085, P = .083; AI (n = 5) vs young (n = 5): 68.7 ± 3.0 vs 52.6 ± 5.4, P = .047; AU (n = 5) vs young: 53.4 ± 6.5 vs 52.6 ± 5.4, P = .411) (Figure 4S-U).
Effects of Aging on Brain Expression Levels of Selected Marker Proteins
Results of ELISA analysis revealed a marginally significant increase of proinflammatory IL-1β protein level in the STR of AU but not of AI animals compared to the young control group (n = 5/group; F(2, 12) = 3.127, P = .081; AU vs young: 1.5 ± 0.1 pg/µg vs 1.1 ± 0.1 pg/µg, P = .032; AI vs young: 1.4 ± 0.1 pg/µg vs 1.1 ± 0.1 pg/µg, P = .110) (Figure 5B). In the neocortex and the HC, no differences in IL-1β expression was found between the groups (CTX: F(2, 7) = 0.511, P = 0.621; HC: F(2, 12) = 0.061, P = .941) (Figure 5A and C).
Brain protein expression levels (pg/µg protein) of inflammatory markers, neurotrophic factors, and α7-nAChRs in young, and cognitively unimpaired (AU) or impaired (AI) aged rats. (A-C) Neocortical (nyoung = 4, nAU = 3, nAI = 3), striatal (nyoung = 5, nAU = 5, nAI = 5), and hippocampal (nyoung = 5, nAU = 5, nAI = 5) IL-1β protein levels. (D-F) Neocortical (nyoung = 4, nAU = 3, nAI = 3), striatal (nyoung = 5, nAU = 5, nAI = 5), and hippocampal (nyoung = 5, nAU = 5, nAI = 5) MIP-1α protein levels. (G-I) Neocortical (nyoung = 4, nAU = 3, nAI = 3), striatal (nyoung = 4, nAU = 6, nAI = 4), and hippocampal (nyoung = 5, nAU = 6, nAI = 5) CX3CL1 protein levels. (J-L) Neocortical (nyoung = 4, nAU = 3, nAI = 3), striatal (nyoung = 4, nAU = 6, nAI = 4), and hippocampal (nyoung = 5, nAU = 6, nAI = 5) ICAM-1 protein levels. (M-O) Neocortical (nyoung = 4, nAU = 3, nAI = 3), striatal (nyoung = 5, nAU = 5, nAI = 5), and hippocampal (nyoung = 5, nAU = 5, nAI = 5) CNTF protein levels. (P-R) Neocortical (nyoung = 4, nAU = 3, nAI = 3), striatal (nyoung = 5, nAU = 5, nAI = 5), and hippocampal (nyoung = 5, nAU = 5, nAI = 5) BDNF protein levels. (S-U) Neocortical (nyoung = 4, nAU = 3, nAI = 3), striatal (nyoung = 5, nAU = 5, nAI = 5), and hippocampal (nyoung = 5, nAU = 5, nAI = 5) α7 nAChR protein levels. Asterisks mark significant changes in protein expression levels compared to young animals: ** P < 0.01, * P < 0.05 (univariate ANOVA + post hoc LSD). α7 nAChR, α7 nicotinic acetylcholine receptors; BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; ICAM-1, intercellular adhesion molecule 1; IL-1β, interleukin-1β; MIP-1α, macrophage inflammatory protein 1α.
Surprisingly, neither the AU nor AI groups showed a significant change in the expression of the selected chemokines MIP-1α and CX3CL1 in any of the tested brain regions (MIP-1α: CTX: F(2, 7) = 0.652, P = .550; STR: F(2, 12) = 0.051, P = .950; HC: F(2, 12) = 0.173, P = .843; CX3CL1: CTX: F(2, 7) = 1.156, P = .368; STR: F(2, 11) = 0.122, P = .886; HC: F(2, 13) = 0.012, P = .988) (Figure 5D-I).
The adhesion molecule ICAM-1 protein expression levels did not differ between the groups in the STR and in the HC (STR: F(2, 11) = 0.463, P = .641; HC: F(2, 13) = 0.764; P = .486) (Figure 5K and L). However, in the neocortex, ICAM-1 protein levels were significantly higher in AU animals compared to young rats (CTX: F(2, 7) = 12.837, P = .005; AU (n = 3) vs young (n = 4): 255.2 ± 27.9 pg/µg vs 171.9 ± 5.3 pg/µg, P = .006) (Figure 5J).
In contrast with mRNA expression levels, protein levels of hippocampal CNTF were significantly decreased in the AI group compared to the young group (n = 5/group; F(2, 12) = 5.386, P = .021; AI (n = 5) vs young (n = 5): 0.6 ± 0.1 pg/µg vs 1.3 ± 0.2 pg/µg, P = 0.007) (Figure 5O). In addition, there was a (non-significant) tendency to decreased CNTF protein expression levels in the HC of the AU compared to the young group (AU (n = 5) vs young (n = 5): 0.9 ± 0.1 pg/µg vs 1.3 ± 0.2 pg/µg, P = 0.070) (Figure 5O). In the neocortex and the STR, no significant main effect of aging could be observed on CNTF protein levels of rats (CTX: F(2, 7) = 2.298, P = 0.171; STR: F(2, 12) = 2.167, P = 0.151) (Figure 5M and N). Aging caused no significant main effect on neocortical, striatal, or hippocampal BDNF levels (CTX: F(2, 7) = 1.311, P = .328; STR F(2, 12) = 1.159, P = .347; HC: F(2, 12) = 0.458; P = 0.643) (Figure 5P-R).
Similarly, there was no significant main effect on the protein expression level of α7 nAChR in any of the examined brain areas (CTX: F(2, 7) = 1.882, P = 0.222; STR: F(2, 12) = 2.120, P = 0.163; HC: F(2, 12) = 0.383, P = 0.690) (Figure 5S-U). However, in the STR, pairwise comparisons showed a (non-significant) tendency of upregulation of α7 nAChR in the AU group compared to the young group (AU (n = 5) vs Young (n = 5): 2.1 ± 0.1 pg/µg vs 1.6 ± 0.2 pg/µg, P = 0.081) (Figure 5T).
DISCUSSION
The present study provides evidence for the beneficial procognitive effects of combination treatments in the naturally aged rats using memantine and an α7 nAChR-selective agent (PHA-543613). First, we confirmed that aged rats display substantial cognitive impairment compared to young rats, which is in line with several earlier preclinical studies.35-38 Monotreatments with memantine or PHA-543613 alleviated age-related cognitive impairments of rats at certain doses. Although the DI was not significantly increased by memantine, comparison of the time spent with the exploration of the novel vs the familiar objects showed that memantine at both the lowest (0.1 mg/kg) and highest (1.0 mg/kg) doses successfully restored recognition memory of aged rats. PHA-543613 exerted its maximum efficacy at the lowest 0.3 mg/kg dose following the usually observed pattern of cholinergic modulation which resembles an inverted U-shaped function.39 Although the 5 mg/kg memantine dose can be considered therapeutically relevant for rats,40 there is supporting evidence for the effectiveness of memantine at 1.0 mg/kg or even much lower doses without undesirable behavioral side-effects. For example, Wise and Lichtman demonstrated that memantine used at or below 1.0 mg/kg dose improved the spatial memory of rats in the radial arm maze.41 In line with that, we have earlier reported that memantine, administered in fairly low doses (0.1 to 1 mg/kg) effectively improved the long-term spatial memory performance of rats in the Morris Water Maze Task24 or reversed scopolamine-induced short-term memory deficit of rats in the spontaneous alternation task.23 We also found that the effective dose of PHA-543613 in the spontaneous alternation task was at 3 mg/kg.10 Surprisingly, in the present experiments, a 10-fold lower dose of PHA-543613 (0.3 mg/kg) already successfully ameliorated the natural cognitive deficits of aged rats. Other selective α7 nAChR agonists, for example, AZD0328, also improved NOR performance of mice at ultra-low doses (0.00178-1.78 mg/kg).42,43 The differences found between the optimal doses of PHA-543613 in the different studies may be—at least partially—explained by the differences in the applied cognitive impairment models, which suggests that much more relevant preclinical data can be obtained using the naturally aging animals compared to pharmacological dementia models.
Next, we aimed to investigate the effects of co-administration of subeffective doses of memantine (0.01 mg/kg) and PHA-543613 (0.1 mg/kg). Here, we found that the long-term recognition memory impairment in aged rats was effectively reversed by the memantine-PHA-543613 combination treatment showing a superior cognitive enhancer effect of the combination treatment over corresponding monotreatments. The present results indicate a most likely positive interaction between memantine and α7 nAChR agonists, and suggest the central role of α7 nAChRs in the observed interaction between memantine and PHA-543613, which has also been previously corroborated by several preclinical studies. For example, Nikiforuk et al. demonstrated that higher cognitive flexibility and enhanced recognition memory were observed in rats when memantine was combined with galantamine or selective α7 nAChR PAMs (CCMI or PNU-120596).44 In the same study, the effects of combination treatments were successfully blocked by the α7 nAChR antagonist methyllycaconitine (MLA), indicating that cognitive enhancement by the combination treatment was indeed an α7 nAChR-dependent process. These results are further supported by the study of Busquet et al., showing that spontaneous alternation and object recognition of mice were facilitated by combining subeffective doses of galantamine and memantine.21 In addition, we have previously demonstrated that a considerable improvement of spatial short-term (working) and long-term memory may also be achieved with the combination treatments using PHA-543613 and memantine.23,24
The exact mechanism underlying the beneficial interaction between α7 nAChR agents and memantine has not been clarified, yet. An in vitro study using mouse brain slices showed that memantine in fact binds to α7 nAChRs with higher affinity compared to NMDA receptors, exerting an antagonistic effect on α7 nAChRs, too.26 In addition, MLA has been demonstrated to improve memory acquisition in rats at low doses, suggesting a cognitive effect that is overall similar to that of the agonists of the α7 nAChR.45 Accordingly, it can be concluded that the direct antagonist action of memantine on α7 nAChRs may also be involved in the superior procognitive effects of the combination treatments, especially at low doses. Thus, α7 nAChRs may serve as the common target of memantine and PHA-543613 in their memory improvement action.
As it has been recently described, α7 nAChRs act as key regulators of cholinergic anti-inflammatory responses, which may also relate to memory processes.46,47 Based on these, agents acting on α7 nAChRs may be beneficial in the treatments of inflammatory diseases such as AD, via their anti-inflammatory effects.48 Foucault-Fruchard et al. recently confirmed the anti-inflammatory properties of PHA-543613 through the potential modulation of microglial activation in an in vivo excitotoxic rat model.49 In agreement with this, following LPS administration, PNU-120596 was shown to inhibit the activation markers of microglia and astrocytes and the release of proinflammatory cytokines such as IL-1β and TNF-α in the HC and the cortex.50 Accordingly, such mechanisms may well be involved in the therapeutic efficacy of the α7 nAChR-targeted combination treatments. However, further investigations are needed to provide decisive evidence on the involvement of the α7 nAChR modulation-induced anti-inflammatory responses in the observed cognitive enhancer effects.
As increased levels of neuroinflammation in the aging brain are considered a main pathological hallmark of NCDs,51,52 we evaluated the differences between the neuroinflammatory state of rats. Results indicated that both AU and AI animals exhibited an elevated mRNA level of IL-1β, MIP-1α and ICAM-1. However, the mRNA expression of CX3CL1 was upregulated only in AI rats with no significant difference in the protein expression. Earlier studies did not agree on age-related changes in CX3CL1 expression (for a review see Eugenín et al.).53 Bachstetter et al. 54 found reduced CX3CL1 protein level in aged rats without any differences in the mRNA expression level, while in the study of Lyons et al., both mRNA and protein levels of CX3CL1 were lower in aged rats compared to young adult rats.54,55 Other studies reported the increase of CX3CL1 protein expression in aged rats,56,57 which is in line with the increase of CX3CL1 mRNA expression in aged rats in the present study.
Increased mRNA expression of the presently examined markers was manifested in higher protein levels only in the case of IL-1β in the STR and ICAM-1 in the neocortex of AU rats compared to young rats. Similarly to our present findings, other studies reported higher protein expression of IL-1β and ICAMs in the HC of aged (22 m.o.) rats,28,58,59 suggesting that neuroinflammatory markers can be reliably detected in senescent rats (22 m.o. or older).
Neurotrophic factors such as BDNF and CNTF are known to be neuroprotective since they can promote synaptic plasticity and neuronal survival.60-63 Indeed, in our present study, CNTF protein expression was significantly lower in the HC of aged animals with memory impairment (AI group) compared to young rats. Surprisingly, the cortical and hippocampal CNTF mRNA was upregulated in the AI group which might be explained by a mechanism that aims at the compensation of lower neurotrophic support at the transcriptional level during aging. However, the compensatory mechanism may not be manifested in the protein expression because of yet unknown posttranscriptional or posttranslational mechanisms.
We also found that AI rats exhibited a robust upregulation of α7 nAChR mRNA in all examined brain regions with no significant changes in the α7 nAChR protein expression. Probably, the small sample sizes and the lower sensitivity of the ELISA method compared to RT-PCR may explain why no systematic increase in protein levels could be statistically confirmed as a result of the upregulation of α7 nAChR mRNA in the brain of aged rats. Previous body of evidence in the literature is also ambiguous regarding the direction of change of α7 nAChR expression in the brain during aging. Several studies found that expression of α7 nAChRs markedly decreased in NCDs, mainly in brain areas related to cognitive functions such as the frontal cortex and HC.64-67 However, others reported the age-related upregulation of α7 nAChRs, particularly on the mRNA level, which was also confirmed by our present observations.16,68,69 Nevertheless, a supposed upregulation of α7 nAChRs may also explain the fairly low effective doses of the selective α7 nAChR agonist in aged rats, namely that PHA-543613 ameliorated the cognitive deficit of the aged rats in a 10-fold lower dose in the present study compared to its effects in scopolamine-treated young adult rats in our earlier studies.10,23 An obvious limitation of the biomarker assays in our study was that it only enabled the comparison of the mRNA and protein expression of various markers in AI, AU, and young rats without being able to investigate the effects of the tested pharmacological treatments on the expression of the biomarkers in the different experimental groups, as the presently applied acute within-subject treatment design was not appropriate to effectively and permanently alter biomarker levels in the brain. Similarly, cessation of pharmacological treatments long before brain sampling also made it unlikely to detect possible residual influence of treatments on biomarker expression. A further investigation using a subchronic treatment regime will assess the effects of memantine, PHA-543613, and their combination on the expression of different biomarkers in the brain.
In conclusion, in the present study, we confirmed the beneficial interaction between memantine and the α7 nAChR-selective agonist PHA-543613 in naturally aged rats demonstrating a translationally more relevant cognitive improvement. While the present findings may provide a new strategy for the development of future procognitive therapies, it is important to note that further research involving repeated (subchronic or chronic) treatments is required to confirm the long-term benefits and safety profile of our approach. Based on our present and previous results, we further hypothesize a prominent role of α7 nAChRs in the cognitive enhancer effects of the combination treatments. Therapeutic potential of combination treatments that are based on pharmacological interactions on the α7 nAChR in the brain is also supported by the finding that expression of the α7 nAChRs may become upregulated during aging, allowing a better accessibility to the ligand binding sites and providing a good opportunity for their effective pharmacological modulation.
Acknowledgments
Behavioral experiments were performed in collaboration with the Animal Facility at the Szentágothai Research Centre of the University of Pécs. We are grateful to Kitti Schmeltzer and Gergő Deák for animal care.
The scientific work/research and/or results disclosed in this article were reached with the sponsorship of the Gedeon Richter Talentum Foundation in the framework of the Gedeon Richter Excellence PhD Scholarship of Gedeon Richter Plc.
Author contributions
Nóra Bruszt (Data curation [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Visualization [equal], Writing—original draft [equal]), Zsolt Kristóf Bali (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Writing—review & editing [equal]), Lili Nagy (Data curation [equal], Investigation [equal], Methodology [equal], Writing—review & editing [equal]), Kornélia Bodó (Formal analysis [equal], Investigation [equal], Methodology [equal]), Péter Engelmann (Formal analysis [equal], Investigation [equal], Methodology [equal], Resources [equal]), and István Hernádi (Conceptualization [equal], Funding acquisition [equal], Methodology [equal], Resources [equal], Supervision [equal], Writing—review & editing [equal])
Funding
This work was supported by the UNKP-22-3 New National Excellence Program of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund (to N.B.) and by the National Research, Development and Innovation Fund of the Hungarian Government (grant number “K 129247” to I.H.). The project TKP2021-EGA-10 was implemented with support provided by the National Research Development and Innovation Fund of Hungary financed under the TKP2021-EGA funding scheme (to I.H.). We also appreciate the funding from the Medical School Research Foundation of the University of Pécs (grant number KA-2022-03 to P.E.). This project was supported by the National Research, Development and Innovation Office (PharmaLab, RRF-2.3.1-21-2022-00015).
Conflicts of interest
The authors declare that the research was conducted in the absence of any commercial, non-financial, or financial relationships that could be construed as a potential conflict of interest.
Data availability
Data are available on request.
References
Author notes
Present address: Kornélia Bodó, National Laboratory of Virology, Szentágothai Research Centre, University of Pécs, 20 Ifjúság str., H-7624 Pécs, Hungary
