Tacrolimus Decreases Cognitive Function by Impairing Hippocampal Synaptic Balance: A Possible Role of Klotho

doi:10.21203/rs.3.rs-280191/v1

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Tacrolimus Decreases Cognitive Function by Impairing Hippocampal Synaptic Balance: A Possible Role of Klotho

https://doi.org/10.21203/rs.3.rs-280191/v1

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published 25 Aug, 2021

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The influence of long-term tacrolimus treatment on cognitive function remains to be elucidated. Using chronic tacrolimus neurotoxicity in mice, we evaluated the influence of tacrolimus on cognitive function, synaptic balance, its regulating protein (Klotho), and oxidative stress in the hippocampus. Compared to vehicle-treated mice, tacrolimus-treated mice showed significantly decreased hippocampal-dependent spatial learning and memory function. Furthermore, tacrolimus caused synaptic imbalance as demonstrated by decreased excitatory synapses and increased inhibitory synapses, and downregulated Klotho in a dose-dependent manner; its downregulation was localized to excitatory hippocampal synapses. Moreover, tacrolimus increased oxidative stress and was associated with the activation of the PI3K/AKT pathway in the hippocampus. The present results indicate that tacrolimus impairs cognitive function via synaptic imbalance, and that these processes are associated with Klotho downregulation at synapses through tacrolimus-induced oxidative stress in the hippocampus.

Neurobiology of Disease

Klotho

Cognitive dysfunction

Tacrolimus

Synaptic imbalance

Oxidative stress

Hippocampus

Neurological alteration is a complication in one-third of organ transplant recipients after transplantation [1, 2]. Neurological complications such as cognitive, emotional, and behavioral changes in kidney transplant patients are associated with calcineurin inhibitors (CNIs), particularly chronic tacrolimus (TAC) treatment [3]; however, the causal relationship between TAC and impaired cognitive function remains unclear.

The hippocampus plays a pivotal role in cognitive functions including learning, memory, and spatial orientation [4, 5]. Learning and memory require interaction between new neurons and the hippocampal neural network. This process is based on synaptic plasticity, which is altered in response to synaptic neuronal activity [6].

Klotho, expressed in the brain and kidney, plays roles in anti-aging and cognitive function in the hippocampus and the brain [7, 8]. In animal studies, Klotho deficiency causes cognitive decline [9, 10], whereas Klotho overexpression leads to increased cognitive function [11]. In humans, Klotho levels in the cerebrospinal fluid decrease with aging in Alzheimer's disease [12], whereas Klotho upregulation enhances cognitive functions [11] in healthy individuals.

Moreover, Klotho possesses antioxidant functions and plays an important role in protecting against oxidative stress. Using Klotho^+/− mice, we previously reported that Klotho deficiency renders the kidney more susceptible to TAC-induced oxidative stress [13] and that Klotho treatment inhibits the PI3K/AKT pathway, suggesting its essential role in protecting against CNI-induced oxidative stress. Thus, oxidative stress caused by long-term CNI exposure may affect cognitive function via Klotho in the hippocampus.

Here, to investigate the influence of TAC on cognitive function in the hippocampus, we evaluated hippocampal-dependent spatial memory function and the expression of related synapse markers and neurotransmitter receptors in TAC-treated mice. Furthermore, we examined the spatiotemporal expression of Klotho in the hippocampus of TAC-treated mice.

Animal care and preparation

To examine the effective dose of TAC-induced neurotoxicity, three different doses of TAC (Prograft, Astellas Pharma Inc., Ibaraki, Japan) diluted in olive oil (MilliporeSigma, Billerica, MA, USA) were subcutaneously injected into mice for 4 weeks. After 1-week acclimation, weight-matched mice were randomized into the following four groups (n = 10 per group): (1) vehicle (VH) receiving only olive oil at the same volume as the TAC groups, (2) TAC 1.5 receiving 1.5 mg/kg body weight of TAC in olive oil; (3) TAC 3, 3 mg/kg; (4) TAC 12, 12 mg/kg. The doses and duration of TAC administration were in accordance with our previous studies reporting that TAC causes organ damage including nephrotoxicity and neurotoxicity [14–16]. Further, the TAC concentration used in all experiments compared with the VH group was 3 mg/kg, the effective concentration for TAC-induced neurotoxicity.

Behavior tests

Open-field test

The open-field test (OFT) was performed to evaluate general locomotor activity in mice, as described previously with slight modification [17, 18]. Each mouse (n = 7) was placed in the center area of the OFT apparatus (50 × 50 × 38 cm³), and its motility was observed during a 5-min period. Locomotor activity was measured from the total distance moved (cm), movement time (s), and velocity (cm/s), and was analyzed using a computerized video-tracking system with the SMART program (PanLab Co., Barcelona, Spain). After behavioral monitoring, the apparatus surface was cleaned with 70% ethanol solution and dried before testing the next animal.

Barnes maze test

The Barnes maze (BM) was used with slight modification [19] to evaluate spatial learning and memory functions in mice. To evaluate spatial learning and memory, mice were habituated to the BM through a training session (i.e., learning) for four consecutive days; the probe trial was then performed on day five. Mice were placed in a light-blocked starting box at the center of the platform, which comprised four quadrants (the target quadrant including the escape hole, and the opposite quadrant, representatively) in the apparatus (diameter, 92 cm; height, 100 cm) with 20 holes located at the border. Only one escape hole was opened with a target box that was located under the escape hole, and the other 19 holes were closed. The starting box was removed after 10 s, and the mice were allowed to explore the platform. During 4 d, the mice were trained in spatial acquisition with four trials of 3 min at 20-min intervals. In the acquisition trials, mice that found the escape hole were placed in the target box for 60 s; those that did not find the escape hole for 3 min were gently guided to the target box. The escape latency of these mice was recorded as 180 s. On day 5, a probe trial was conducted for 90 s to evaluate the short-term memory of mice. The memory functions were indicated by the latency time (s) required to reach the escape hole, retention time at each quadrant of the platform, and the number of visits to the target hole. The BM test was performed using a computerized video-tracking system with the SMART program (PanLab Co.).

Tissue preparation

The experimental animals were anesthetized with tiletamine-zolazepam (10 mg/kg, intraperitoneal injection; Zoletil 50, Virbac Laboratories, Carros, France) and xylazine (15 mg/kg, intraperitoneal; Rompun®, Bayer, Leverkusen, Germany), and were then euthanized through transcardial perfusion with a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH = 7.4) for 30 min or through decapitation. For histological evaluation, the brain was post-fixed in 4% paraformaldehyde for 4 h and then embedded in wax. For immunoblotting and reverse transcription-quantitative PCR (RT-qPCR) analyses, the hippocampus was separated and immediately frozen in liquid nitrogen. The samples were stored at -70°C until further use.

Immunofluorescence

Dewaxed 3-µm brain sections were incubated in retrieval solution (pH 6.0) and then washed in phosphate-buffered saline. After blocking with 10% normal donkey serum (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h, the sections were incubated overnight at 4°C with a rabbit polyclonal antibody against Klotho (1:200; ab203576; Abcam, Cambridge, UK). Primary antibody binding was visualized using peroxidase-labeled donkey anti-rabbit IgG (1:100; Jackson ImmunoResearch) and 0.05% 3,3-diaminobenzidine tetrahydrochloride (DAB; Vector Laboratories, Burlingame, CA, USA) with 0.01% H₂O₂ as the substrate. For double or triple immunohistochemistry, the brain sections were incubated at 4°C overnight with the following antibodies: polyclonal guinea pig anti-vGlut1 (1:1000; #AB5905; Millipore Corp., Billerica, Mass., USA), monoclonal mouse antibody anti- PSD95 (1:200; MABN68; Millipore), MAP2 (1:400; M4403; MilliporeSigma), monoclonal rabbit antibody against gephyrin (1:200; AIP-005; Almone Labs, Jerusalem, Israel), β-3 tubulin (1:600; #5568; Cell Signaling Technology Inc., Danvers, MA, USA), polyclonal rabbit anti-Klotho antibody (Abcam) conjugated with biotinylated goat anti-rabbit IgG (BA-1000; Vector Laboratories Inc.), and 8-Hydroxy-2'-deoxyguanosine (1:200; 8-OHdG; MOG-100P; Japan Institute for the Control of Aging, Shizuoka, Japan). Antibody staining was visualized using the following secondary antibodies: Dichlorotriazinylamino fluorescein (DTAF)-conjugated streptavidin (1:100; Jackson ImmunoResearch), Cy3-conjugated goat anti-rabbit antibody (1:1000; Jackson ImmunoResearch), Cy3-conjugated goat anti-mouse antibody (1:1000; Jackson ImmunoResearch), Alexa Fluor 488-conjugated goat anti-guinea pig antibody (1:200; Thermo Fisher, Waltham, MA, USA), Alexa Fluor 488-conjugated goat anti-mouse antibody (1:200; Thermo Fisher), or Alexa Fluor 647-conjugated goat anti-mouse antibody (1:200; Thermo Fisher). Cell nuclei were then counterstained with 4,6-diamidino-2-phenylindole (DAPI; Roche, Mannheim, Germany) for 10 min.

Slides were observed using a confocal microscope (LSM700; Carl Zeiss Co. Ltd., Oberkochen, Germany). Images were converted to the TIFF format, and contrast levels were adjusted using Adobe Photoshop v. 13 (Adobe System, San Jose, CA, USA). The IMARIS program (Bitplane, Zurich, Switzerland) was used for three-dimensional (3D) rendered Z-stacked confocal images.

Immunoelectron microscopic analysis

Small blocks (1–2 mm²) of mouse hippocampal CA1 regions were cryopreserved by immersing in 2.3 M sucrose and were frozen in liquid nitrogen. Frozen tissues were cut with 2-µm-thick semithin cryosections using a glass knife in a Leica EM UC7 ultramicrotome equipped with an FC7 cryochamber (Leica, Wetzlar, Germany).

For pre-embedding immuno-EM, the sections were incubated at 4°C overnight with polyclonal rabbit anti-Klotho antibody (1:200; Abcam). The sections were then labeled with peroxidase-conjugated goat anti-rabbit IgG (1:100; Jackson ImmunoResearch) for immunoperoxidase staining, or with nanogold particle (1 nm)-conjugated anti-rabbit secondary antibody (1:100; Nanoprobes, Stony Brook, NY, USA) for immunogold/silver staining. Each Klotho-labeled tissue was visualized using 0.05% DAB as a chromogen for peroxidase, or a silver enhancement kit (Nanoprobes) for nanogold-conjugated antibodies. After postfixation, dehydration, and embedding in Epon 812 (Polysciences, Warrington, PA, USA), the areas of interest were excised and glued onto resin blocks. Ultra-thin sections (70 nm thick) were cut and observed under an electron microscope (JEM 1010, JEOL, Tokyo, Japan) after uranyl acetate staining.

Correlative EM was performed as previously described [20].

The sections were then labeled with DAPI to counterstain the cell nuclei. The stained sections were covered with coverslips and examined using a confocal microscope for fluorescence. Differential interference contrast settings were used to identify the region of interest in the sections through EM. The coverslips were then removed, and the sections were processed for EM as described above.

Immunoblotting

Frozen hippocampal tissues were homogenized in Pro-Prep Protein Extraction Solution (Intron Biotechnology, Seongnam, South Korea) per the manufacturer’s protocol. Equimolar amounts (20 µg) of protein were separated through SDS-PAGE and electroblotted onto a polyvinylidene fluoride membranes (Millipore). The membranes were incubated overnight at 4°C with the following antibodies: polyclonal rabbit anti-Klotho antibody (1:1000; Abcam), p-AKT (Ser473) (1:1000; #9271S; Cell Signaling Technology Inc.), t-AKT (1:1000; #9272 S; Cell Signaling Technology Inc.), p-FoxO3a (Ser253) (1:1000; #9466 S; Cell Signaling Technology Inc.), t-FoxO3a (1:1000; #2497S; Cell Signaling Technology Inc.), SOD/MnSOD (1:1000; ab13533; Abcam), monoclonal rabbit anti-gephyrin (1:1000; Almone Labs), polyclonal guinea pig anti-vGlut1 (1:1000; #AB5905; Millipore), monoclonal mouse anti-VGAT (1:1000; 131 011; Synaptic System), PSD95 (Millipore), and monoclonal mouse anti-β-actin (1:10000; A5441; Sigma-Aldrich). The immunoreactive bands were detected using a chemiluminescence kit (ATTO Corporation, Tokyo, Japan). Each band was quantified by relative density as a percentage of the ratio of the TAC group to that of the VH group, and each density was normalized to that of β-actin (Quantity One version 4.4.0; Bio-Rad, Hercules, CA, USA).

RT-qPCR

Total RNA from the mouse hippocampus samples was extracted using RNA isolation reagent (RNA-Bee; Tel Test, Inc., TX, USA). The purified RNA (5 µg) was reverse transcribed into first-strand complementary DNA using a Dyne 1st -Strand cDNA Synthesis Kit (DyneBio Inc., Seongnam, Korea). RT-qPCR amplification was conducted using SYBR Green PreMix in a LightCycler 480 system (Roche, Rotkreuz, Switzerland). The mRNA expression level was normalized to that of GAPDH using the change in the cycle threshold method. The following primers were used for qPCR: 5′-AAAAGTCGGGGTCTCTCTGAC-3′ and 5′-CAGTCGGTCCAAAATTCTTGTGA-3′ for mouse GABA A receptor subunit alpha 1 (GABARA1), 5′-TTACAGTCCAAGCCGAATGTCCC-3′ and 5′-ACTTCTGAGGTTGTGTAAGCGTAGC-3′ for mouse GABA A receptor subunit alpha 2 (GABARA2), 5′-CAAGAACCTGGGGACTTTGTGAA-3′ and 5′-AGCCGATCCAAGATTCTAGTGAA-3′ for mouse GABA A receptor subunit alpha 3 (GABARA3), 5′-GAGACTGGTGGATTTTCCTATGG-3′ and 5′-GGTCCAGGTGTAGATCATCTCACT-3′ for mouse GABA A receptor subunit alpha 4 (GABARA4), 5′-CCCTCCTTGTCTTCTGTATTTCC-3′ and 5′-TGATGTTGTCATTGGTCTCGTCT-3′ for mouse GABA A receptor subunit alpha 5 (GABARA5), 5′-ATCTGCCTGGTTCCATGATGT-3′ and 5′-AGCCATAGCTCTCTAGGTCCA-3′ for mouse GABA A receptor subunit delta (GABARD), 5′-AGAAAAACCCTCTTCTTCGGATG-3′ and 5′-GTGGCATTGTTCATTTGAATGGT-3′ for mouse GABA A receptor subunit gamma 2 (GABARG2), 5′-CTCGCCCTTGTCGTACCAC-3′ and 5′-GTCCGCCCTGAGAAATCCAG-3′ for mouse AMPA receptor subunit 1 (GluR1), 5′-GTGTCGCCCATCGAAAGTG-3′ and 5′-AGTAGGCATACTTCCCTTTGGAT-3′ for mouse AMPA receptor subunit 2 (GluR2), 5′-ATCGGATATTCGCAAGGAACC-3′ and 5′-CCATAGGGCCAGATTCCACA-3′ for mouse kainate receptor subunit 1 (GluR6), 5′-AAAGGCCAGAGGTCCAACTAT-3′ and 5′-CCCCTTCAGCATTAAGTATGGGT-3′ for mouse kainate receptor subunit 4 (KAR1), 5′-ATAGTCGCCTTCGCCAATCC-3′ and 5′-GTGTCCGTGGTCTCGTACTG-3′ for mouse kainate receptor subunit 5 (KAR2), 5′-AGAGCCCGACCCTAAAAAGAA-3′ and 5′-CCCTCCTCCCTCTCAATAGC-3′ for mouse NMDA receptor subunit 1 (NR1), 5′-TGATGAACCGCACTGACCCTA-3′ and 5′-TGGGGATGAAAGTCTGTGAGG-3′ for mouse NMDA receptor subunit alpha 2 (NR2A), 5′-GCCATGAACGAGACTGACCC-3′ and 5′-GCTTCCTGGTCCGTGTCATC-3′ for mouse NMDA receptor subunit beta 2 (NR2B), 5′-AGGTCCTAATGTCACTGACTCTC-3′ and 5′-GCCATAAAGGGTCCTATCAGAC-3′ for mouse metabolic glutamate receptor subunit 7 (mGluR7), and 5′-AGGTCGGTGTGAACGGATTTG-3′ and 5′-TGTAGACCATGTAGTTGAGGTCA-3′ for mouse Gapdh. All experiments were performed in triplicate on samples from individual mice (n = 4).

Quantitative and statistical analysis

Klotho-labeling profiles and other synapse markers were evaluated by counting approximately 20 randomly selected areas (50 × 50 µm² per field) in each stained tissue section at ×400 magnification using a color image analyzer (TDI Scope Eye, version 3.0, for Windows).

Differences with P-values less than 0.05 were considered significant. All statistical analyses were conducted using GraphPad Prism version 5 (GraphPad Software Inc., San Diego, CA, USA).

Effects of TAC treatment on cognitive function in mice

We performed behavioral analysis to evaluate whether TAC impairs cognitive function in mice. We used TAC at 3 mg/kg body weight as previously reported [14–16]. First, locomotor activity in VH and TAC groups was examined using the OFT. There was no significant difference in the total distance moved, movement time, and velocity, indicating similar locomotion between the two groups (Fig. 1A).

Spatial learning performance and memory were then examined using the BM. During the four acquisition trial days, the latency time to the escape hole was gradually decreased in both groups; however, the TAC group showed longer latency time than that of the VH group (Fig. 1B) (day 1, 81.1 ± 4 s for VH, 86.7 ± 1.4 s for TAC; day 2, 42.9 ± 3.9 s for VH, 60.3 ± 6.7 s for TAC; day 3, 31.2 ± 2.4 s for VH, 57.6 ± 6.7 s for TAC; day 4, 23.1 ± 0.8 s for VH, 46.9 ± 3.2 s for TAC). On day 5, the probe trial day, TAC-treated mice spent less time in the target quadrant (56.9 ± 2.8 % for VH; 21.4 ± 3.4 % for TAC) and more time in the opposite quadrant compared (14.7 ± 3.2 % for VH; 36.2 ± 6.6 % for TAC) to VH mice (Fig. 1C); further, the TAC group showed increased primary latency time (22.7 ± 1.5 s for VH; 45.9 ± 3.5 s for TAC) and distance of movement, and a decreased number of visits to the escape hole (10.3 ± 2.2 for VH; 5.7 ± 0.7 for TAC) compared with the VH group (Fig. 1D–F). These findings suggest that TAC treatment significantly influenced cognitive function in mice, resulting in impaired spatial learning and memory.

Effects of TAC treatment on synapses

First, we investigated the effects of TAC treatment on synaptic changes in the mouse hippocampus, using excitatory- (vGlut1 and PSD95; Fig. 2A, C, E) and inhibitory synaptic markers (VGAT and gephyrin; Fig. 2B, D, F), respectively. TAC-treated mice showed decreased immunoreactive puncta for excitatory synapse markers vGlut1 and PSD95 (Fig. 2A) (vGlut1, 160 ± 5.5 for VH, 91.3 ± 4.4 for TAC; PSD95, 758.2 ± 17.7 for VH, 371.8 ± 14.9 for TAC); vGlut1/PSD95 co-labeled puncta were also decreased by TAC treatment (Fig. 2C) (307.8 ± 19.6 for VH, 125.4 ± 12.1 for TAC). In contrast, immunoreactive puncta for inhibitory synapse markers VGAT and gephyrin were increased upon TAC treatment (Fig. 2B) (VGAT, 31.3 ± 2.1 for VH, 63.8 ± 2.9 for TAC; gephyrin, 85.4 ± 6.9 for VH, 148.6 ± 9.9 for TAC), and VGAT/gephyrin co-labeled puncta were decreased (Fig. 2D) (11.6 ± 1.3 for VH, 48 ± 3.9 for TAC).

On immunoblotting for excitatory synapse markers vGlut1 and PSD95 (Fig. 2E) and inhibitory synapse markers VGAT and gephyrin (Fig. 2F), the immunoreactive bands of each synapse marker were quantified and compared in both VH and TAC groups. The labeling intensity of excitatory synapses was remarkably decreased, whereas inhibitory synapses ere increased in the TAC group compared to the VH group. Furthermore, RT-qPCR analysis revealed that glutamate receptors were significantly downregulated, whereas GABAergic receptors were upregulated in the TAC group compared to the VH group (Fig. 3).

Influence of TAC treatment on Klotho expression in hippocampus

As TAC treatment impaired cognitive function in mice, we examined the changes in Klotho. First, we evaluated the distribution of Klotho expression and the influence of TAC on hippocampal Klotho expression. Klotho was expressed in the pyramidal neurons in the hippocampal CA1 region on immunohistochemistry (Fig. 4A), consistent with a previous report [21]. Immunoblotting for hippocampal proteins revealed that the Klotho antibody was detected as a single band at ~ 130 kDa in both VH and TAC groups (Fig. 4B). The labeling intensity of Klotho in the hippocampus was remarkably decreased in a dose-dependent manner (61.1 ± 4.6 % for TAC 1.5; 52.2 ± 8.6 % for TAC 3; 43.7 ± 4.8 % for TAC 12) in the TAC-treated groups compared with that of the VH group (100 ± 4.3 %).

Second, we identified the phenotype of Klotho expression in the CA1 region, a particularly vulnerable area in the hippocampus (boxed area in Fig. 1A), and evaluated the influence of TAC on Klotho expression in neuronal cells. Triple labeling was performed using Klotho and the neuronal markers, β-III tubulin and microtubule-associated protein 2 (MAP2). Most Klotho expression in the pyramidal cell layer (pcl) was detected in preserved neurons showing β-III tubulin and MAP2 immunoreactivity with neuronal processes elongated to the stratum radiatum (sr) (Fig. 4C − D). Prominent neuronal Klotho expression, including the soma and neurites, was observed in the VH group (Fig. 4C and E − H), whereas the TAC group showed weak Klotho immunoreactivity in the neurons (Fig. 4D, and I − L).

Influence of TAC treatment on Klotho expression in neuronal dendrites

As shown in Fig. 4E and I, Klotho immunoreactivity was observed in neuronal processes and neurites. We then performed double labeling for Klotho and MAP2, a marker specific for neuronal dendrites. MAP2-positive dendrites showed prominent Klotho expression with punctate structures along the dendritic processes (Fig. 5A, C). In particular, Klotho was localized along the outer part of the dendrite, indicating presumptive synapse structures, identified through 3D reconstruction (Fig. 5B, D) of their morphology and topographical distribution. Quantitative analysis revealed significantly lesser Klotho-expressing puncta in the TAC group than in the VH group (Fig. 5E).

Localization of Klotho in neuronal dendrites

Klotho expression in MAP2-immunoreactive neuronal dendrites was first observed through confocal microscopy (Fig. 6A − C), and the same sections from these images were subjected to electron microscopy (Fig. 6D − E). Electron microscopy revealed presynaptic vesicles (white asterisks in Fig. 6F) and adjacent postsynaptic density (PSD) (arrowheads in Fig. 6F), an electron-dense structure at the postsynaptic membrane [22]. Klotho immunoreactivity indicated by electron-dense DAB grains (black asterisks in Fig. 6G) or silver-enhanced gold particles (arrows in Fig. 6H) was revealed in pre-embedding immunoelectron microscopic images. Reactive Klotho was detected in the postsynaptic profiles including the PSD (arrowheads in Fig. 6G − H), and composed the junction along with the vesicle-containing presynapses (white asterisks in Fig. 6G − H).

Effects of TAC treatment on synaptic Klotho expression

Furthermore, we investigated the involvement of Klotho in inhibitory and excitatory synapses after TAC treatment. As Klotho is localized in postsynapses, we performed double labeling for Klotho and an excitatory postsynaptic marker (PSD95; Fig. 7A-F) or for Klotho and an inhibitory postsynaptic marker (gephyrin; Fig. 7H-M). Confocal microscopic images (Fig. 7A − B, D − E, H − I, K − L) of reactive Klotho-, PSD95-, and gephyrin-positive puncta were 3D reconstructed (Fig. 7C, F, J, M) using IMARIS and the number of Klotho-expressing synapses in total synapses was quantified. Klotho/PSD95 co-labeled puncta were lesser after TAC treatment (224.8 ± 5.4 for VH, 88 ± 2.2 for TAC); the rate of double-labeled expression in total PSD95-positive excitatory postsynapses was also decreased significantly (0.3 ± 0.005 for VH, 0.24 ± 0.006 for TAC) (Fig. 7G). In contrast, Klotho/gephyrin co-labeled puncta were increased (64.3 ± 5.6 for VH, 106.6 ± 3.5 for TAC) in TAC-treated mice; however, the proportion of double-labeled expression in total gephyrin-positive inhibitory postsynapses was retained in both groups (0.77 ± 0.05 for VH, 0.75 ± 0.02 for TAC) (Fig. 7M).

Effects of TAC treatment on oxidative stress in the hippocampus

To investigate whether Klotho downregulation is associated with oxidative stress regulation after TAC treatment, we double labeling for Klotho and 8-OHdG, a marker of cellular oxidative DNA damage, in the hippocampus. The nuclear immunoreactivity of 8-OHdG in weak Klotho-expressing neurons was remarkably greater in the TAC group than in the VH group (Fig. 8A − H). Further, since the antioxidant effects of Klotho are regulated by MnSOD signaling [23–25], we examined the increased oxidative stress in the hippocampus of TAC-treated mice through immunoblotting for AKT, FoxO3a, and MnSOD (Fig. 8I–J). Consistent with Klotho downregulation in TAC-treated mice, p-AKT and p-FoxO3a were upregulated, whereas MnSOD, a FoxOs target gene, was downregulated.

Our results clearly indicate that long-term TAC treatment impairs cognitive function. Using an experimental model of chronic TAC neurotoxicity, we confirmed that chronic TAC treatment in mice caused cognitive dysfunction and impaired spatial learning and memory function. TAC treatment caused synaptic imbalance and downregulated Klotho in the hippocampus. These findings indicate that TAC decreases cognitive function by impairing hippocampal synaptic plasticity and that Klotho is involved in this process.

Hippocampal function is based on synapses that regulate the excitation-inhibition balance; the excitatory-to-inhibitory input ratio remains constant and is conserved under normal conditions [26–31]. We examined synaptic changes in the mouse hippocampus using the excitatory and inhibitory synaptic markers. TAC treatment decreased the number of excitatory synapses and increased the number of inhibitory synapses. These changes were also observed at the protein and mRNA levels. These findings suggest that TAC causes synaptic imbalance in the hippocampus.

Klotho plays an important role in cognitive function in the hippocampus [8, 10, 11, 32, 33]. However, the localization of Klotho in the synapse remains unclear. Thus, we evaluated the distribution of Klotho expression in the hippocampus. On examining the hippocampal CA1 region, identifying the phenotype of Klotho-expressing cells, and examining the subcellular localization of Klotho, we found that Klotho was expressed in neuronal processes and was localized to the postsynaptic neurites at the subcellular level.

Next, the influence of TAC on the changes in Klotho were examined. TAC downregulated hippocampal Klotho in a dose-dependent manner. As previously reported [21], Klotho expression was localized in hippocampal neurons, and TAC treatment downregulated Klotho in neurons. Further, TAC treatment downregulated Klotho at excitatory but not inhibitory synapses. Thus, TAC downregulates Klotho at the synapses in hippocampal neurons.

Overall, these findings suggest that TAC causes synaptic imbalance in the hippocampus, and Klotho downregulation is potentially involved in this process. Thus far, the effect of Klotho on synaptic function remains unclear. We thus speculate that Klotho regulates the balance between input and output signals, and Klotho downregulation at excitatory synapses results in synaptic imbalance. Overall, we propose that TAC-induced impairment of cognitive function is associated with synaptic imbalance via Klotho downregulation at hippocampal excitatory synapses.

Using Klotho^+/− mice, we previously reported that Klotho deficiency renders the kidney more susceptible to TAC-induced injury, which is closely associated with aggravated TAC-induced oxidative stress [13, 34]. This study investigated whether TAC-induced oxidative stress affects hippocampal Klotho expression. Klotho is an important regulator of oxidative stress, and its effect is associated with the PI3K-Akt-FoxOs signaling pathway [9, 13, 35–37]. Our results indicate that TAC increased 8-OHdG, an oxidative damage marker, activated PI3K/AKT-mediated phosphorylation of FoxO3a, and inhibited FoxO3a binding to the MnSOD promoter, leading to reduced MnSOD expression (Fig. 7I-J). Thus, TAC-induced oxidative stress downregulated Klotho via PI3K/AKT pathway activation in the hippocampus.

CNI treatment is associated with neurotoxicity [38]. However, the influence of CNIs on cognitive function remains controversial [39–43], possibly owing to the diverse etiology of cognitive impairment after organ transplantation, including age underlying disease. Our study clearly defines the association between CNI and cognitive function. Impaired cognitive function in organ transplant recipients results in hindrances in daily living (including medication adherence) and increased morbidity and mortality. However, the importance of cognitive function has been overlooked thus far. Our study indicates the importance of evaluating cognitive function in organ transplant recipients with long-term exposure to CNIs.

Our study defines the association between Klotho expression and TAC-induced cognitive dysfunction; however, it did not include a functional study. Nonetheless, it suggests that decreased cognitive function by TAC is causally associated with Klotho downregulation in the hippocampus. Thus, further studies using Klotho-deficient or -overexpressing mice are needed to determine the role of Klotho at the synaptic level.

In summary, our data indicate that TAC treatment impairs cognitive function via synaptic imbalance in the hippocampus, and that these processes may be associated with Klotho downregulation through TAC-induced oxidative stress (Fig. 9).

Authors’ Contributions

YJ Shin, SW Lim, and CW Yang; designed the research and wrote the report; YJ Shin, and C Sheng: conducted the animal experiments; EJ Ko, TR Riew, and HL Kim: performed the histological experiments; YJ Shin, BH Chung, MY Lee, and CW Yang; analyzed the data and edited the manuscript.

Funding

This work was supported by the Korean Health Technology R&D Project, Ministry for Health & Welfare, Republic of Korea (HI14C3417), and the Basic Science Research Program through a National Research Foundation of Korea (NRF) grant funded by the Ministry of Education (NRF-2018R1D1A1B07044219) and the Ministry of Science, ICT & Future Planning (NRF- 2020R1A2C2012711).

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors of this manuscript have no conflicts of interests.

Ethical Approval

This study was approved by the Institutional Animal Care and Use Committee (IACUC) at the School of Medicine, Catholic University of Korea (CUMC-2020-0110-02).

Consent to Participate

Not applicable

Consent for Publication

Not applicable

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Tacrolimus Decreases Cognitive Function by Impairing Hippocampal Synaptic Balance: A Possible Role of Klotho

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Journal Publication

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Abstract

Figures

Introduction

Materials And Methods

Animal care and preparation

Behavior tests

Open-field test

Barnes maze test

Tissue preparation

Immunofluorescence

Immunoelectron microscopic analysis

Immunoblotting

RT-qPCR

Quantitative and statistical analysis

Results

Effects of TAC treatment on cognitive function in mice

Effects of TAC treatment on synapses

Influence of TAC treatment on Klotho expression in hippocampus

Influence of TAC treatment on Klotho expression in neuronal dendrites

Localization of Klotho in neuronal dendrites

Effects of TAC treatment on synaptic Klotho expression

Effects of TAC treatment on oxidative stress in the hippocampus

Discussion

Declarations

References

Status:

Journal Publication

Version 1