I₂ imidazoline receptor modulation protects aged SAMP8 mice against cognitive decline by suppressing the calcineurin pathway

Abstract

Brain aging and dementia are current problems that must be solved. The levels of imidazoline 2 receptors (I₂-IRs) are increased in the brain in Alzheimer’s disease (AD) and other neurodegenerative diseases. We tested the action of the specific and selective I₂-IR ligand B06 in a mouse model of accelerated aging and AD, the senescence-accelerated mouse prone 8 (SAMP8) model. Oral administration of B06 for 4 weeks improved SAMP8 mouse behavior and cognition and reduced AD hallmarks, oxidative stress, and apoptotic and neuroinflammation markers. Likewise, B06 regulated glial excitatory amino acid transporter 2 and N-methyl-d aspartate 2A and 2B receptor subunit protein levels. Calcineurin (CaN) is a phosphatase that controls the phosphorylation levels of cAMP response element-binding (CREB), apoptotic mediator BCL-2-associated agonist of cell death (BAD) and GSK3β, among other molecules. Interestingly, B06 was able to reduce the levels of the CaN active form (CaN A). Likewise, CREB phosphorylation, BAD gene expression, and other factors were modified after B06 treatment. Moreover, phosphorylation of a target of CaN, nuclear factor of activated T-cells, cytoplasmic 1 (NFAT_C1), was increased in B06-treated mice, impeding the transcription of genes related to neuroinflammation and neural plasticity. In summary, this I₂ imidazoline ligand can exert its beneficial effects on age-related conditions by modulating CaN pathway action and affecting several molecular pathways, playing a neuroprotective role in SAMP8 mice.

Electronic supplementary material

The online version of this article (10.1007/s11357-020-00281-2) contains supplementary material, which is available to authorized users.

Introduction

Aging has become a problem worldwide, since older people are more prone to developing chronic and degenerative diseases. At the brain level, aging affects several molecular pathways that predispose patients to neurodegeneration, causing dementia, cognitive impairment and degraded quality of life. Among dementias, the most prevalent is Alzheimer’s disease (AD) [23]. AD has aroused considerable interest because of its strong influence on quality of life among elderly individuals and because of the limited drugs available to combat cognitive loss and neuropsychiatric symptoms.

β-Amyloid deposition in senile plaques and tau hyperphosphorylation forming neurofibrillary tangles are specific hallmarks of AD [50]. However, there are no successful pharmacological treatments that modify the progression of AD, given that acetylcholinesterase inhibitors and memantine fail to stop the progression of dementia [16, 17]. Apart from the use of approved drugs, several clinical attempts have been made to treat AD progression using various other strategies, such as immunotherapy against β-amyloid and beta-secretase (BACE) inhibitor administration, but the results have been disappointing [15]. These results show that addressing only the “β-amyloid cascade hypothesis” cannot fully control the progression of the disease, and this hypothesis also cannot explain the advanced neuronal damage in AD. Therefore, identifying new pharmacological targets for AD treatment is an active area of research.

In most neurodegenerative processes, including AD, neuroinflammation and oxidative stress (OS) are common traits. It is well-accepted that Ca²⁺ dysfunction is a consequence of homeostatic imbalance in nerve cells that unleashes a string of molecular and cellular processes, including neuroinflammation, OS, changes in neuronal plasticity, differential expression of glutamate and cholinergic receptors, and amyloid pathology [52]. Together, these processes end with cognitive decline and neurodegeneration. Calcineurin (CaN), also known as protein phosphatase 2B, is a Ca²⁺-dependent Ser/Thr phosphatase that is highly abundant in the brain, appearing at high levels in neurons and low levels in glia in healthy adult animals [31]. CaN is related to long-term potentiation (LTP) and long-term depression (LTD), and dysregulation of CaN has been linked with cognitive loss in an AD mouse model [6, 45]. Of importance, CaN levels and signaling are increased in the cortex in AD patients [63] and in the contexts of other human neurodegenerative pathologies, including Parkinson’s disease (PD) [12], Lewy body aggregation [35] and vascular pathology [44]. Moreover, CaN activity prevents fear memory formation in the amygdala by dephosphorylation and inhibition of downstream kinases, including AKT and extracellular signal-regulated kinase (ERK) [33]. N-methyl-d-aspartate receptor (NMDAR) [39, 60] and glycogen synthase kinase 3β (GSK3β) [61] are some of the key actors in central nervous system function that are controlled by the phosphatase activity of CaN, which in turn is controlled by calcium calmodulin kinase II (CaMKII) and intracellular Ca²⁺ levels [7]. Nuclear factor of activated T-cells (NFAT) consists of at least two different components, one with nuclear localization and one that is phosphorylated and localized in the cytoplasm [27]. Furthermore, recently, the CaN pathway has been observed to link astrocytic Ca²⁺ dysregulation to neuroinflammation, glutamate, β-amyloid accumulation and synaptotoxicity [52].

Imidazoline 2 receptors (I₂-IRs) [8] are increased in AD brains [22, 47], and radioactive ligands have been studied as biomarkers for AD and PD progression in patients [58, 62]. There is evidence that I₂-IR ligands reduce neurodegenerative processes, including cognitive decline, neuroinflammation, OS and AD hallmarks, but less is known about the upstream mechanism involved in the beneficial effects of I₂-IR modulation. Thus, the objective of this work was to delineate the molecular mechanisms involved in the neuroprotective effect of I₂-IR modulation in a mouse model of AD linked to the aging process, the senescence-accelerated mouse prone 8 (SAMP8) model. To this end, we used a newly synthesized I₂-IR ligand, diethyl (1RS,3aSR,6aSR)-5-(3-chloro-4-fluorophenyl)-4,6-dioxo-1-phenyl-1,3a,4,5,6,6a-hexahydropyrrolo[3,4-_c]pyrrole-1-phosphonate, named B06, which has outstanding affinity and selectivity for I₂-IRs over α₂ adrenoreceptors [3, 18].

The SAMP8 strain is a non-transgenic mouse strain established through phenotypic selection of the AKR/J mouse strain, and is an attractive model with which to study aging processes, especially age-related deterioration of learning and memory, emotional disorders and neurochemical alterations [43, 57]. At approximately 5 months of age, the mice begin to undergo an accelerated process of senescence, and the brain aging manifests as severe cognitive decline and neuroinflammation [4]. It is considered a late-onset AD mouse model characterized by altered amyloid precursor protein (APP) processing and high levels of tau hyperphosphorylation [11, 37]. Moreover, inflammatory and OS markers are present at early ages and during adulthood [24, 25].

Methods

In vivo studies in mice

Twelve-month-old female SAMP8 mice (n = 23) (Envigo, Sant Feliu de Codines, Barcelona, Spain) were used to carry out cognitive and molecular analyses. The animals were randomly allocated to two experimental groups: the SAMP8 control group (control) (n = 12), which was administered vehicle (2-hydroxypropyl)-β-cyclodextrin 1.8% in drinking water, and the SAMP8 group, which was treated with the I₂-IR ligand B06 (5 mg/kg) (n = 11). The animals had free access to food and water and were kept under standard temperature conditions (22 ± 2 °C) and 12 h/12 h light/dark cycles (300 lx/0 lx). B06 (5 mg/kg/day) was diluted in 1.8% (2-hydroxypropyl)-β-cyclodextrin and administered through drinking water. After 4 weeks of treatment, behavioral and cognitive tests, including short- and long-term memory, were performed to study the effects of treatment on learning and memory. Weight and water consumption were controlled each week, and the B06 concentration was adjusted accordingly to reach the optimal dose until euthanasia.

The studies and procedures for the mouse behavior tests, brain dissection and extractions followed the ARRIVE and standard ethical guidelines (European Communities Council Directive 2010/63/EU and Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research, National Research Council 2003) and were approved by the bioethical committees of the University of Barcelona and the Government of Catalonia. All efforts were made to minimize the number of animals used and their suffering.

Novel object recognition test (NORT)

Briefly, mice were placed in a 90° two-arm 25-cm-long, 20-cm-high, 5-cm-wide black maze. Before performing the test, the mice were individually habituated to the apparatus for 10-min periods for 3 days. On day 4, the animals were allowed to freely explore in a 10 min acquisition trial (first trial), for which they were placed in the maze in the presence of two identical objects at the end of each arm (Fig. 1a). After a delay (2 h for short-term memory evaluation and 24 h for long-term evaluation), the animal was allowed to explore the old object and one novel object in each trial (Fig. 1a). The time that the mice spent exploring the novel object (TN) and the time that the mice spent exploring the old object (TO) were measured. A discrimination index (DI) was defined as (TN − TO)/(TN + TO). Exploration of an object was defined as pointing the nose toward the object at a distance ≤2 cm and/or touching it with the nose [5]. Turning or sitting around the object was not considered exploration. To avoid object preference biases, the objects were counterbalanced.

Fig. 1.

Scheme for NORT (a) and OFT (b) experimental paradigms. The I₂-IR ligand improved the novel object recognition abilities [measured as discrimination index, (DI)] in SAMP8 treated with B06 at 5 mg/Kg/day (B06 5 mg/kg) in comparison with the SAMP8 control in both summary short-term memory (c) and summary long-term memory (d). In the open field test (OFT), 12-month-old SAMP8 treated with B06 at 5 mg/Kg/day (B06 5 mg/kg) presented a significant increase in the distance traveled (e), the percentage of time spent in the center zone (f) and the number of rearings (g). Values represented are mean ± standard error of the mean (SEM); n = 15 (control n = 8, B06 n = 7); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs. control

Open field test (OFT)

The open field test (OFT) was performed as previously described [24] (Fig. 1b). Briefly, mice were placed at the center of and allowed to explore a white plywood box (50 × 50 × 25 cm) for 5 min. Behavior was scored with SMART^® [Spatial Monitoring and Reporting Tool] version 3.0 software, and each trial was recorded for later analysis. The parameters scored included the center stay duration, number of rearings, number of defecations and distance traveled.

Determination of oxidative stress

Hydrogen peroxide (H₂O₂) was measured as an indicator of OS, and it was quantified using a hydrogen peroxide assay kit (Cat. No. MAK165, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions.

Immunodetection experiments

Brain processing

Three days after the behavioral and cognitive tests, mice were euthanized for protein extraction and RNA and DNA isolation. After euthanasia, the brains were immediately removed from the skulls, and the hippocampi were dissected, frozen and maintained at −80 °C.

For an IHC experiment, mice were anesthetized (ketamine 100 mg/kg and xylazine 10 mg/kg, intraperitoneally) and then perfused intracardially with 4% paraformaldehyde (PFA) diluted in 0.1 M phosphate buffer solution. Their brains were removed and postfixed in 4% PFA overnight at 4 °C. Afterwards, the solutions were changed to PFA + 15% sucrose. Finally, the brains were frozen on powdered dry ice and stored at −80 °C until sectioning.

Protein level determination by western blotting

For subcellular fractionation, 150 μL of buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA pH 8, 0.1 mM EGTA pH 8, 1 mM DTT, 1 mM PMSF, protease inhibitors) was added to each sample, and the mixtures were incubated on ice for 15 min. Next, the samples were homogenized with a tissue homogenizer, 12.5 μL Igepal 1% was added, and the Eppendorf tubes were vortexed for 15 s. Following 30 s of full-speed centrifugation at 4 °C, the supernatants (cytoplasmic fractions) were collected, 80 μL of buffer C (20 mM HEPES pH 7.9, 0,4 M NaCl, 1 mM EDTA pH 8, 0.1 mM EGTA pH 8, 20% glycerol 1 mM DTT, 1 mM PMSF, protease inhibitors) was added to each pellet, and the pellets were incubated under agitation at 4 °C for 15 min. Subsequently, the samples were centrifuged for 10 min at full speed at 4 °C. The supernatants (nuclear fractions) were collected.

For western blotting (WB), aliquots of 20 μg of hippocampal protein were used. Protein samples from mice were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8–12%) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). Afterwards, the membranes were blocked in 5% nonfat milk in 0.1% Tris-buffered saline with Tween 20 (TBS-T) for 1 h at room temperature before being incubated overnight at 4 °C with the primary antibodies listed in Supplementary Table 1.

The membranes were washed and incubated with secondary antibodies for 1 h at room temperature. Immunoreactive proteins were viewed with a chemiluminescence-based detection kit following the manufacturer’s protocol (ECL Kit; Millipore, Burlington, MA, USA), and digital images were acquired using a ChemiDoc XRS+ System (Bio-Rad, Hercules, CA, USA). Semiquantitative analyses were carried out using Image Lab software (Bio-Rad), and the results are expressed in arbitrary units (AU), with the control protein levels set as 100%. Protein loading was routinely monitored by immunodetection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin.

Immunofluorescence

Brain coronal sections of 30 μm were obtained (Leica Microsystems CM 3050S cryostat, Wetzlar, Germany) and kept in a cryoprotectant solution at −20 °C until use. Free-floating slices were placed in a 24-well plate and washed with 0.1M PBS. Next, the free-floating sections were blocked with a solution containing 1% (BSA), 0,3% Triton X-100, 0.1M PBS for 20 min at room temperature; washed with PBS 0.1M two times for 5 min each; and incubated with the primary antibodies listed in Supplementary Table 2 overnight at 4 °C. On the following day, the coronal slices were washed with 0.1M PBS 0.1M 2 times for 5 min each and then incubated with the secondary antibodies listed in Supplementary Table 2 at room temperature for 1 h. Later, the sections were washed two times for 5 min each with 0.1M PBS and were incubated with 5μM Hoechst staining solution (Sigma-Aldrich, St. Louis, MO) for 5 min in the dark at room temperature. Finally, the slices were mounted using Fluoromount-G (EMS, Hatfield, Pennsylvania, USA), and image acquisition was performed with a fluorescence laser microscope (Olympus BX41, Hamburg, Germany) by using x4 and x20 magnification. At least four images from 4 different individuals in each group were analyzed with ImageJ/Fiji software available online from the National Institutes of Health.

RNA extraction and gene expression determination

Total RNA isolation was carried out using TRIzol^® reagent according to the manufacturer’s instructions. The yield, purity and quality of RNA were determined spectrophotometrically with a NanoDrop™ ND-1000 apparatus (Thermo Scientific, Waltham, MA, USA) and an Agilent 2100B Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA samples with 260/280 ratios and RNA integrity numbers (RINs) higher than 1.9 and 7.5, respectively, were selected. Reverse transcription-polymerase chain reaction (RT-PCR) was performed. Briefly, 2 μg of messenger RNA (mRNA) was reverse-transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA).

SYBR^® Green real-time PCR was performed on a StepOnePlus Detection System (Applied Biosystems) with SYBR^® Green PCR master mix (Applied Biosystems). Each reaction mixture contained 6.75 μL of complementary DNA (cDNA) (with a concentration of 2 μg), 0.75 μL of each primer (with a concentration of 100 nM) and 6.75 μL of SYBR^® Green PCR master mix (2×).

TaqMan-based real-time PCR (Applied Biosystems) was also performed in a StepOnePlus Detection System (Applied Biosystems). Each 20 μL TaqMan reaction contained 9 μL of cDNA (25 ng), 1 μL of 20× TaqMan gene expression assay probe and 10 μL of 2× TaqMan universal PCR master mix.

The data were analyzed utilizing the comparative cycle threshold (Ct) (ΔΔCt) method, in which the levels of a housekeeping gene are used to normalize differences in sample loading and preparation. Normalization of expression levels was performed with β-actin for SYBR^® Green-based real-time PCR and Gapdh for TaqMan-based real-time PCR. The primer sequences and TaqMan probes used in this study are presented in Supplementary Table 3. Each sample was analyzed in duplicate, and the results represent the n-fold differences in the transcript levels among different groups.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism version 8 statistical software. The data are expressed as the mean ± standard error of the mean (SEM). Means were compared with two-tailed Student’s t test. Statistical significance was considered when p values were <0.05. Statistical outliers were determined with Grubbs’ test and when necessary were removed from the analyses.

Results

Prevention of memory loss and behavioral impairment in SAMP8 mice after I₂-IR ligand treatment

The NORT demonstrated significant differences between the control and I₂-IR ligand B06 groups in both short- and long-term evaluations. Significantly higher DI values were obtained for the B06-treated mice than for the control mice at 2 h and 24 h after novel object exposure, indicating a neuroprotective action of B06 against the characteristic SAMP8 mouse memory loss (Fig. 1c–d).

In addition, the results regarding locomotor activity, time spent in the center area and number of rearings, as assessed with the OFT paradigm, revealed significant changes in behavior in the B06 group in comparison with the control group (Fig. 1e–g).

AD hallmark modifications in the hippocampi of SAMP8 mice induced by I₂-IR ligand treatment

The levels of key proteins involved in APP processing were evaluated. The I₂-IR ligand B06 promoted significant increases in soluble APPα (sAPPα) levels but clearly tended to decrease soluble APPβ (sAPPβ) levels (Fig. 2a–b). Accordingly, the gene expression of Adam10, a constitutive α-secretase, increased, indicating a shift to the non-amyloidogenic pathway (Fig. 2c). Moreover, the gene expression of both insulin-degrading enzyme (Ide) and neprilysin (Nep) was increased after B06 treatment (Fig. 2c).

Fig. 2.

Treatment with the I₂-IR ligand B06 resulted in significant differences in the amyloid processing and Αβ degradation pathway between the 12-month-old control SAMP8 (Control) and the SAMP8 treated with B06 at 5 mg/Kg/day (B06 5 mg/kg). Representative western blot for sAPPα and sAPPβ protein levels and quantification (a, b). Values in bar graphs were adjusted to 100% for the protein of control SAMP8 (Control). Representative gene expression for Adam10, Ide and Nep (c). Gene expression levels were determined by real-time PCR. Values are the mean ± standard error of the mean (SEM); (n = 3–5 animals per group); *p < 0.05; **p < 0.01 vs. Control

Tau hyperphosphorylation is a characteristic posttranslational modification in aged SAMP8 mice. B06 treatment induced significant decreases in phosphorylation at the Ser404 and Ser396 sites in tau protein (Fig. 3a–b). There are two main kinases implicated in tau hyperphosphorylation: glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase 5 (CDK5). GSK3β phosphorylated at Ser9 is the inactive form of the enzyme and is correlated with reduced tau phosphorylation. As expected, the I₂-IR ligand B06 increased p-GSK3β (Ser9) levels, indicating that it reduced kinase activity (Fig. 3c). CDK5 is also activated by phosphorylation, and p25, a coactivator, controls its activity. The results showed that the I₂-IR ligand-treated group presented decreases in the p-CDK5 level and p25/p35 ratio (Fig. 3d–e).

Fig. 3.

The I₂-IR treatment mediated a significant decrease in tau phosphorylation and the implicated kinases in 12-month-old SAMP8 treated with B06 at 5 mg/Kg/day (B06 5 mg/kg) when compared to control SAMP8 (Control). Representative western blot for ratio p-Tau (Ser396 and Ser404), ratio p-GSK3β (Ser9), ratio p-CDK5, p25/35 and quantification (a–e). Values in bar graphs were adjusted to 100% for protein of control SAMP8 (Control). Values are the mean ± Standard error of the mean (SEM); (n = 3–5 animals per group); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs. Control

I₂-IR ligand treatment changes synaptic and apoptotic markers in SAMP8 mice

The I₂-IR ligand B06 reduced the protein levels of the NMDA2B receptor, increased those of the form phosphorylated at Tyr1472 and increased those of the NMDA2A receptor significantly (Fig. 4a–c).

Fig. 4.

Changes in NMDARs, neuronal plasticity and kinase pathways induced by I₂-IR ligand B06 in 12-month-old SAMP8 after treatment at 5 mg/Kg/day (B06 5 mg/kg) in comparison with 12-month-old control SAMP8 (Control). Representative western blot for NMDAR2B, NMDAR2A, ratio p-NMDAR2B (Tyr1472). Ratio p-CREB in nucleus protein levels and quantification (a–d). Representative western blot for kinases PKA, ratio p-AKT and quantification (e, f). Values in bar graphs were adjusted to 100% for the protein of control SAMP8 (Control). Representative gene expression for Bdnf (g). Gene expression levels were determined by real-time PCR. Values are the mean ± standard error of the mean (SEM); (n = 3–5 animals per group); *p < 0.05; **p < 0.01; ***p < 0.001 vs. Control

The levels of calcium/calmodulin-dependent protein kinase II (CaMKII), a marker of synaptic plasticity, did not show significant changes, but the levels of the phosphorylated form of cAMP response element-binding protein (CREB) were dramatically increased (Fig. 4d) in the B06 group. Accordingly, the gene expression of the CREB target brain-derived neurotrophic factor (Bdnf) was increased in the B06 group (Fig. 4g).

Protein kinase A (PKA) is a master regulator of the activity of CREB, among other transcription factors. B06-treated animals showed increased protein levels of PKA α (the catalytic fragment) (Fig. 4e). We found significant recovery of AKT, also known as protein kinase B, phosphorylation and subsequent activation (Fig. 4f) in the B06 group, indicating a pathway of neuroprotective regulation after B06 treatment.

B-cell lymphoma 2 (BCL-2), Bax, BCL-2-associated agonist of cell death (BAD) and caspase 3 are key factors in apoptotic signaling in neurons. B06 was able to reduce caspase 3 and Bcl-2-like protein 4 (Bax) protein levels; surprisingly, it also reduced BCL-2 protein levels (Fig. 5a–c). An increase in p-BAD was also observed (Fig. 5d); however, in this case, phosphorylation of BAD indicated a lack of capacity to form apoptotic pores by dimerization of BAD, which subsequently weakened the proapoptotic role of this factor. Overall, prevention of apoptotic mechanisms followed treatment with the I₂-IR ligand B06.

Fig. 5.

Treatment with the I₂-IR ligand B06 suppressed apoptosis by inhibiting the implicated apoptotic factors in 12-month-old SAMP8 treated with B06 at 5 mg/Kg/day (B06 5 mg/kg) as compared to control SAMP8 (Control). Representative western blot for caspase-3, Bax, BCL-2, ratio p-BAD (Ser136) and quantification (a–d). Values in bar graphs were adjusted to 100% for the protein of control SAMP8 (Control). Values are the mean ± standard error of the mean (SEM); (n = 3–5 animals per group); *p < 0.05; **p < 0.01 vs. Control

Neuroinflammation and oxidative state changes in SAMP8 mice after I₂-IR ligand treatment

A highly significant decrease in GFAP protein levels was observed in the B06 group (Fig. 6a), indicating that astrogliosis and neuroinflammation processes were ameliorated in I₂-IR ligand B06-treated mice. Astrocytes control glutamatergic signaling through glutamate transporters, and B06 was able to enhance the protein levels of excitatory amino acid transporter (EAAT) 2 (Fig. 6b). The expression of proinflammatory cytokines including interleukin 6 (Il-6), Il-18, Il-1β, interferon gamma (Ifn-γ), tumor necrosis factor-alpha (Tnf-α) and C-X-C motif chemokine ligand 10 (Cxcl-10) was decreased after treatment with the I₂-IR ligand B06 (Fig. 6c), and the decrease reached significance for Il-6, Il-18 and Il-1β. H₂O₂ levels in the hippocampus were significantly diminished in the B06 mouse group, showing that global redox homeostasis was shifted due to the antioxidant role of the I₂-IR ligand in SAMP8 mice (Fig. 6d). The expression of nuclear factor-erythroid 2-related factor 1 (Nrf1), a key gene controlling the oxidative cell environment, was higher in the group treated with the I₂-IR ligand B06 than in the untreated mouse group (Fig. 6e). In addition, the gene expression of antioxidant machinery enzymes such as hemoxygenase 1 (Hmox 1) was increased, whereas that of aldehyde dehydrogenase 2 (Aldh2) was reduced, indicating that B06 prevented the SAMP8 brain from experiencing an oxidant environment by neutralizing radical oxygen species (ROS) (Fig. 6e). Conversely, a significant increase in the gene expression of inducible nitric oxide synthase (iNOS) was found, although this increase could have improved synaptic function (Fig. 6e). Finally, immunostaining quantification of GFAP fluorescence intensity demonstrated that B06 treatment significantly reduced GFAP staining, especially in the dentate gyrus (DG) and CA1 regions (Fig. 6f–m), suggesting a reduction in astrogliosis. Moreover, immunostaining quantification of S100A9 fluorescence intensity showed that B06 treatment reduced S100A9 staining, especially in the CA1 and CA3 regions, but the reductions were not significant (Fig. 6f–m).

Fig. 6.

I₂-IR ligand, B06, attenuated neuroinflammation and OS state in 12-month-old SAMP8 treated mice at 5 mg/Kg/day (B06 5 mg/kg) when compared to the control SAMP8 (Control). Representative western blot for GFAP, GLT-1/EAAT-2 (a–b). Representative images for GFAP (c) and S100A9 immunostaining (c) and quantification for GFAP and S100A9 on the bar chart (d–j). Representative gene expression for inflammatory markers Il-6, Il-18, Il-1β, Ifn-γ, Tnf-α and Cxcl-10 (k) and OS markers Hmox1, iNOS, Nrf1 and Aldh2 (l). Quantification of intracellular H₂O₂ (μM) (m). Gene expression levels were determined by real-time PCR. Values in bar graphs were adjusted to 100% for the protein of control SAMP8 (Control). DG: dentate gyrus. Scale bar for immunohistochemical images is 200 μm. Values are the mean ± standard error of the mean (SEM); (n = 3–5 animals per group); *p < 0.05; ***p < 0.001 vs. Control

I₂-IR ligand treatment modifies CaN/NFAT signaling in the SAMP8 mouse hippocampus

In light of the obtained results, we focused on CaN, an upstream protein with phosphatase activity toward CREB or BAD that plays a role in neurodegeneration. The protein levels of CaN A, the active form, were reduced after treatment with the I₂-IR ligand B06 (Fig. 7a). We also evaluated NFAT_C1, a different target of CaN. The results showed an increase in the phosphorylated form (Fig. 7b).

Fig. 7.

Modulation of CaN signaling after B06 treatment in 12-month-old SAMP8 treated mice at 5 mg/Kg/day (B06 5 mg/kg). Representative western blot for CaN A, ratio p-NFATc1/NFATc1 and quantification (a, b). Values in bar graphs were adjusted to 100% for protein of control SAMP8 (Control). Values are the mean ± standard error of the mean (SEM); (n = 3–5 animals per group); *p < 0.05; **p < 0.01 vs. Control

As a summary of the results, Fig. 8 shows the molecular alterations related to cognitive improvement as well as the key role of CaN in controlling the cellular response after treatment with the I₂-IR ligand B06.

Fig. 8.

Graphical abstract showing molecular changes in CaN signaling after treatment with B06

Discussion

Here, we report that treatment with the I₂-IR ligand B06 in a SAMP8 mouse model, a model of neurodegeneration linked to aging that is considered to assimilate late-onset AD, has beneficial effects via modulation of the CaN pathway. Imidazoline receptors were described in the 1990s, and I₂-IRs are related to neurodegenerative diseases such as AD [22], Huntington’s disease [46] and PD [46, 58, 62]. However, the signal transduction pathway for I₂-IR remains elusive [8]. Previous reports have indicated putative roles related to monoamine oxidase (MAO) A or B [36] and intracellular calcium concentration control through NMDA receptors or intracellular calcium stores [28, 67]. We recently demonstrated that ligands for I₂-IRs are able to prevent neurodegeneration by acting on the apoptotic mechanism [2] and decreasing the activity of kinases (CDK5, GSK3β, etc.) [1, 26], leading to the recovery of cognitive capabilities in an AD mouse model [26]. However, the intrinsic mechanisms that induce these changes are not precisely known.

B06 is a new improved I₂-IR ligand with a lower Ki for I₂-IR than previous ligands and high selectivity for I₂-IRs over α₂ adrenoceptors [18]. The latter characteristic is of the utmost importance for avoidance of undesirable adverse effects on, for example, the vascular system. We previously reported that administration of B06 to a 5xFAD mouse model, a transgenic representative model of AD, reduces cognitive decline, neuroinflammation, tau hyperphosphorylation and APP processing [18].

In the present work, we demonstrated that the I₂-IR ligand B06 was able to improve cognition and ameliorate anxiety-like behavior in aged SAMP8 mice. Furthermore, we confirmed that on the molecular level, treatment with B06 reduced the exhibition of AD hallmarks such as APP processing and tau hyperphosphorylation, inhibited tau kinase (CDK5 and GSK3β) activation, reduced the gene expression of neuroinflammation markers including Il-6, Il-18 and Tnf-α, and decreased OS.

When the apoptotic pathway was studied, decreases in caspase 3, Bax and BCL-2 levels were found. However, there has been a lack of consistency among I₂-IR studies regarding the reduction in apoptotic signaling [21]. Our results are consistent with those of several studies showing that administration of I₂-IR ligands such as 2-BFI and BU224 can reduce apoptotic marker levels in the rat brain cortex [32]. Considering that I₂-IRs have been reported to be involved in key pathways associated with neurodegeneration, we also evaluated several master pathways in B06-treated senescent mice, including those that are under the control of cytosolic calcium, astrocyte activation and synaptic neural plasticity. The localization of I₂-IRs remains elusive, but several studies have reported astrocytes as a major cell type with I₂-IR binding sites [14]. Of note, astrogliosis and activated microglial cells are associated with amyloid processing, indicating that this AD hallmark is a major trigger of gliosis [59]. After B06 treatment, a very significant decrease in the expression of the hippocampal pan-astrocytic reactive marker GFAP indicated strong control of neuroinflammation and a reduction in astrogliosis that in turn could prevent neuronal function loss. Moreover, S100A9, a Ca²⁺-binding protein with a critical role in modulating the inflammatory response and inducing cytokine release by astrocytes [60], was used as a marker of neuroinflammation mediated by reactive astrocytes. In our study, we found clear reductions in two hippocampal areas, CA1 and CA3, confirming a reduction in the inflammatory state after treatment with B06. Likewise, the expression of the EAAT2 isoform (or Glt 1), a glutamate transporter located predominantly in astrocytes, was increased after treatment with the I₂-IR ligand B06. EAAT2 is implicated in glutamate clearance and has a leading role in the removal of excess glutamate and other potentially toxic mediators [19]. In line with these findings, our previous results for two other I₂-IR ligands [26] showed the same action regarding astrogliosis. However, in contrast, another study on the I₂-IR ligand LSL60101 showed induction of reactive astrocytosis in the facial motor nuclei of neonate rats after short-term treatment [13], suggesting that the effects differ depending on both the physicochemical properties of the I₂-IR ligand and the experimental model.

Notably, in astrocytes, increased CaN activity can lead to modification of the kinase activity of GSK3β [61]. Calcium entry through NMDA2B receptors enhances the activation of GSK3β through CaN phosphatase activity, and in turn, GSK3β amplifies this phosphatase activity, dephosphorylating CREB [56, 60]. In addition, the interaction of I₂-IR ligands with NMDA receptors has been well described [41, 42]. Thus, our results support the idea that modulation of I₂-IRs by B06 is able to induce changes in NMDA receptors. We definitively observed changes in NMDA receptor subunit composition and activation. On the one hand, increases in NMDA2A receptor protein levels were observed. On the other hand, decreases in NMDA2B receptor protein levels with increased phosphorylation were observed. These changes are associated with LTP, which may partially explain the improvement in cognition observed in B06-treated SAMP8 mice.

To further elucidate the molecular mechanisms modulated by B06, we examined the negative crosstalk between AKT and GSK3β signaling that participates in synaptic plasticity [9] controlled by CaN phosphatase activity. As mentioned, B06 treatment reduced GSK3β activation by increasing the levels of its inactive form phosphorylated at Ser9, whereas it activated AKT signaling. Because AKT is a recognized pro-survival molecule that participates in neural plasticity, modulation of AKT signaling in animals treated with the I₂-IR ligand B06 likely contributed to the favorable effects on cognition observed in SAMP8 mice [55, 68].

p-CREB controls the expression of genes related to synaptic disruption and LTP, such as Bdnf [10]. Neuronal growth and survival require the expression of CREB target genes that control various proteins, including BDNF and its receptor tropomyosin-related kinase B (TrkB) [66]. On the one hand, treatment with the I₂-IR ligand B06 increased nuclear p-CREB levels and increased Bdnf gene expression. On the other hand, B06 increased the levels of PKA, which can drive p-CREB nuclear translocation. Of note, PKA acts as a negative modulator of NFATc₁, a transcription factor that regulates the transcription of genes that play crucial roles in axonal outgrowth control [51]. Interestingly, I₂-IR ligand treatment induced an increase in NFATc₁ phosphorylation in parallel with decreases in Il-6, Ifn-γ and Tnf-α gene expression. NFATc₁ and CaN are master regulators that control EAAT2 up- or downregulation [54]. We hypothesize that the observed changes in NFATc₁ are responsible for the increase in EAAT2 described above.

NFATc₁ is dephosphorylated by CaN, which enables its nuclear translocation. Continuous NFAT activation and nuclear signaling result in neurodegenerative morphological abnormalities, including neuritic dystrophy, dendritic spine loss and modulation of β-amyloid accumulation. Indeed, NFAT activity stimulates the amyloidogenic pathway [29, 53] and its inhibition has been found to significantly reduce β-amyloid plaque formation in an AD mouse model [20]. Therefore, a reduction in nuclear NFAT should have beneficial effects in senescence models, in which overactivation of neurodegenerative pathways is a key cause of cognitive decline [24, 25].

The last finding, closely linked with the findings described above, is the implication of CaN in the beneficial effects of the I₂-IR ligand B06 in SAMP8 mice. CaN is a multicomponent protein in which CaN A has phosphatase activity regulated by calcium levels [48]. Calcium dysregulation can be induced by age-related changes, such as OS and inflammation [19, 45]. Furthermore, inhibition of CaN signaling produces neuroprotection in models of injury and disease [40, 64], reduces neuroinflammation [19] and cognitive impairment [34], and improves synapse function [30]. Consistent with these findings, we hypothesized that in SAMP8 mice, which are characterized by neuroinflammation and OS, an imbalance in calcium levels occurs, activating CaN A and inducing neurodegeneration. Specifically, astrocytic CaN is activated under inflammatory conditions and can, for example, activate GSK3β and inactivate AKT, influencing NMDAR-mediated axonal outgrowth [60]. As stated above, the I₂-IR ligand B06 reduced CaN A protein levels, and accordingly, we found activation of AKT and strong inactivation of GSK3β.

Regarding the OS observations, CaN can be activated after H₂O₂ addition to neuronal cultures [49]. Likewise, the reductions in OS markers after treatment with the I₂-IR ligand B06 could have also contributed to a reduction in CaN activity. These findings correlate with cognitive improvement, increasing neuroprotective signaling and reducing tau hyperphosphorylation. Conversely, CaN A can dephosphorylate tau. However, the balance between tau phosphorylation and dephosphorylation is due to a shift in tau kinase activity [65]. Hyperactivation of the PP1A domain of CaN A results in dephosphorylation of a few transcription factors, including CREB (which blocks CREB translocation to the nucleus) and NFAT (which enables NFAT translocation to the nucleus). In both cases, the reduced synaptic and growth gene transcription necessitates plasticity, and the increased expression of proinflammatory factors participates in neurodegenerative processes. Moreover, hyperactivation of the phosphatase 2B (PP2B) domain increases BAD dephosphorylation, favoring the action of BAD as a proapoptotic factor [38].

In conclusion, the data from our study demonstrate that modulation of I₂-IRs by B06 reduces neuroinflammation, OS and CaN protein levels in SAMP8 mice. The decreases in CaN protein levels can explain the changes in CREB, NFAT_c1 and BAD phosphorylation levels. In addition, the decreases in CaN levels result in modification of the kinase activity of GSK3β and AKT, among other molecules, leading to reduced tau hyperphosphorylation and preventing cognitive decline in SAMP8 mice. Collectively, our findings provide evidence that the CaN pathway is a critical component of the neuroprotective effects of I₂-IR ligands on SAMP8 model mice, providing insight into several molecular modifications observed after I₂-IR ligand treatment. In the future, deeper knowledge of the role of the I₂-IR signaling cascade in AD will provide new therapeutic targets for cognitive decline and AD.

Abbreviations

AD

Alzheimer’s disease

Aldh2

Aldehyde dehydrogenase 2

APP

Amyloid precursor protein

BAD

BCL-2-Associated agonist of cell death

Bdnf

Brain-derived neurotrophic factor

CaMKII

Calcium calmodulin kinase II

CaN

Calcineurin

CDK5

Cyclin-dependent kinase

cDNA

Complementary DNA

CREB

cAMP response element-binding

Ct

Cycle threshold

Cxcl-10

C-X-C motif chemokine ligand 10

(EAAT)2

Excitatory amino acid transporter 2

ERK

Extracellular signal-regulated kinase

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

GFAP

Glial fibrillary acid protein

GSK3β

Glycogen synthase kinase 3β

H₂O₂

Hydrogen peroxide

Hmox 1

Hemoxygenase 1

I₂-IR

Imidazoline 2 receptors

Ide

Insulin-degrading enzyme

Ifn-γ

Interferon gamma

iNOS

Inducible nitric oxide synthase

LTD

Long-term depression

LTP

Long-term potentiation

MAO

Monoamine oxidase

mRNA

Messenger RNA

Nep

Neprilysin

NFATc1

Nuclear factor of activated T-cells, cytoplasmic 1

NMDA

N-Methyl-d-aspartate

NMDAR

N-Methyl-d-aspartate receptor

NORT

Novel object recognition test

Nrf1

Nuclear factor-erythroid 2-related factor 1

OFT

Open field test

OS

Oxidative stress

p-Tau

Hyperphosphorylated tau

PD

Parkinson’s disease

PKA

Protein kinase A

PP2B

Phosphatase 2B

PVDF

Polyvinylidene difluoride

ROS

Reactive oxygen species

qPCR

Real-time quantitative PCR

RT-PCR

Reverse transcription-polymerase chain reaction

SAMP8

Senescence-accelerated mouse prone 8

sAPPα

Soluble APP α

sAPPβ

Soluble APP β

SDS-PAGE

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

SEM

Standard error of the mean

TBS-T

Tween 20 TBS

TBS

Tris-buffered saline

TN

Novel object, new location

Tnf-α

Tumor necrosis factor alpha

TO

Old object, old location

TrkB

Tropomyosin-related kinase B

WB

Western blotting

ΔΔCt

Cycle threshold method

Author contributions

CGF, MP and CE contributed to conceptualization and funding acquisition. SA, SRA and AB synthesized and purified B06. CGF and FV performed experiments and formal data analysis. CGF, CE, FV and MP wrote, reviewed and edited the manuscript. All authors read and approved the final version of the manuscript.

Compliance with ethical standards

Declarations

Not applicable.

Competing interests

The authors have no competing interests to declare.