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

The influence of long-term tacrolimus treatment on cognitive function remains to be elucidated. Using a murine model of chronic tacrolimus neurotoxicity, we evaluated the effects 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; the downregulation of Klotho was localized to excitatory hippocampal synapses. Moreover, tacrolimus increased oxidative stress and was associated with activation of the PI3K/AKT pathway in the hippocampus. These 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.


Introduction
Neurological alteration is a post-transplantation complication in one-third of organ transplant recipients [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 [15,16]. 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 [17].
The oxidative stress caused by ROS is regarded as a common pathway of CNI-induced organ injury [18]; however, the influence of CNI-induced oxidative stress on the brain remains to be elucidated. It is well known that the brain is susceptible to oxidative stress, and that the hippocampal neurons are the most vulnerable to oxidative stress in the brain [19]. Therefore, it is presumable that long-term CNI treatment may cause neurotoxicity by inducing oxidative stress in the brain.
KLOTHO is a well-known anti-aging gene [20,21]; its expression was reported in the brain and kidney [22]. KLOTHO encodes a single-pass transmembrane protein that functions as a co-receptor of fibroblast growth factor (FGF)-23 with FGF-R [20]. The extracellular domain of the Klotho protein is cleaved on the cell surface by membrane-anchored proteases and is released into blood [23][24][25], urine [26][27][28], and the cerebrospinal fluid [29]. Klotho is involved in phosphate metabolism and the maintenance of calcium and vitamin D homeostasis [30][31][32]. In addition, it plays a critical role in cognitive function in the brain [22,33]. In animal studies, Klotho deficiency causes cognitive decline [20,34], whereas Klotho overexpression leads to increased cognitive function [35]. In humans, Klotho levels in the cerebrospinal fluid decrease with aging in Alzheimer's disease [36], whereas Klotho upregulation enhances cognitive functions [35] in healthy individuals.
Here, to investigate the influence of TAC on cognitive function in the hippocampus, we evaluated hippocampaldependent 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 hippocampi of TAC-treated mice.

Animal Care and Preparation
All animal procedures and experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the School of Medicine, Catholic University of Korea (CUMC-2020-0110-02), and conducted in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experiments. Eight-week-old male BALB/c mice (Orient Bio, Seongnam, South Korea), initially weighing 25-30 g, were housed with five animals/ cage (Nalge, Rochester, NY, USA) under controlled conditions of temperature (20-26 °C), humidity (50 ± 10%), and light (12-h light-dark cycle) at the animal care facility of the Catholic University of Korea.
To examine the effective dose of TAC-induced neurotoxicity, three different doses of TAC (Prograf, Astellas Pharma, Inc., Ibaraki, Japan) diluted in olive oil (Millipore-Sigma, Billerica, MA, USA) were subcutaneously injected into mice for 4 weeks. After 1-week acclimation, weightmatched 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; and (4) TAC 12, 12vmg/kg. Among the three different doses of TAC, we selected 3 mg/kg for the induction of neurological pathogenic alteration in mice, based on the previous reports that this dose of TAC causes organ toxicity [38,[48][49][50][51][52].

Open Field Test
The open field test (OFT) was performed to evaluate general locomotor activity in mice, as described previously, with slight modifications [53,54]. Each mouse (n = 7) was placed in the center area of the OFT apparatus (50 × 50 × 38 cm 3 ), 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 [55], 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 5. 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 the 4-day learning period, the mice were trained in spatial acquisition with four trials of 3 min each 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
Tissue preparation was performed after the behavior test on the same day. Following the behavior test, 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). For histological evaluation, the mice were euthanized through transcardial perfusion with a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH = 7.4) for 30 min, and the brain was postfixed in 4% paraformaldehyde for 4 h followed by embedded in wax. For immunoblotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses, the mice were euthanized through decapitation, and the hippocampus was separated followed by immediate freezing in liquid nitrogen. The samples were stored at − 70 °C until further use.

Immunoelectron Microscopic Analysis
Small blocks (1-2 mm 2 ) of mouse hippocampal CA1 regions were cryopreserved by immersing in 2.3 M sucrose and frozen in liquid nitrogen. Frozen tissues were cut into 2-µm-thick semithin cryosections using a glass knife in a Leica EM UC7 ultramicrotome equipped with an FC7 cryochamber (Leica, Wetzlar, Germany).
Correlative EM was performed as previously described [56]. Semithin cryosections were incubated with a mixture of polyclonal rabbit anti-Klotho antibody (1:200; Abcam) and monoclonal mouse antibody anti-MAP2 (1:400; Mil-liporeSigma), followed by Alexa Fluor 488-conjugated goat anti-rabbit antibody (1:200; Thermo Fisher) and Cy3-conjugated goat anti-mouse (1:1000; Jackson ImmunoResearch). 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. 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 an 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 1 st -Strand cDNA Synthesis Kit (Dyne Bio, Inc., Seongnam, Korea). RT-qPCR amplification was performed with SYBR Green PreMix using a LightCycler 480 system (Roche, Rotkreuz, Switzerland). The messenger RNA (mRNA) expression levels were normalized to those of GAPDH using the change in the cycle threshold method. The following primers were used for RT-qPCR:

Quantitative and Statistical Analysis
Klotho-labeling profiles and other synapse markers were evaluated by counting approximately 20 randomly selected areas (50 × 50 µm 2 per field) in each stained tissue section at × 400 magnification using a color image analyzer (TDI Scope Eye, version 3.0, for Windows). All data are presented as mean ± standard error; unpaired t tests or one-way ANOVA followed by the Bonferroni post hoc test were used for comparing among groups. Differences with P values less than 0.05 were considered significant. All statistical analyses Fig. 1 Chronic TAC treatment affects spatial learning performance and memory in mice. a Distance moved (cm), movement time (s), and velocity (cm/s) were examined in the open-field box for assessing locomotor activity. No difference in locomotion was observed between the VH and TAC-treated mice. b Total latency time required to find the target hole during the training sessions (days 1-4). c-f During the probe trial (day 5), spatial memory was evaluated by measuring the time spent in the target and opposite quadrants (c), the primary latency (d), the distance moved (e), and the number of visits to the target hole (f). e Representative images showing the tracing path of VH-and TAC-treated mice in the Barnes maze. The TAC group showed significant impairment of spatial learning and memory compared with the VH group. The values shown are mean ± SE (n = 7). *P < 0.05 vs. VH 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. First, locomotor activity in the VH and TAC groups was examined using the OFT. There was no significant difference in the total distance moved, movement time, and velocity, which indicated similar locomotion between the two groups (Fig. 1a).
Spatial learning performance and memory were then examined using the BM. During the 4-day acquisition trial, the latency time to the escape hole gradually decreased in both groups; however, the TAC group showed a longer latency time than the VH group (Fig. 1b (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 suggested that TAC treatment significantly influenced cognitive function in mice, resulting in impaired spatial learning and memory.
After 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 the VH and TAC groups. The labeling intensities of excitatory synapses were remarkably decreased, whereas those of inhibitory synapses were increased in the TAC group compared to those in 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 those in the VH group (Fig. 3). Fig. 2 Effects of TAC on the immunoreactive changes in the excitatory and inhibitory synapses of hippocampal neurons. a, b Expression of excitatory presynaptic and postsynaptic markers (vGlut1 and PSD95, respectively) and inhibitory presynaptic and postsynaptic markers (VGAT and gephyrin, respectively). c, d Quantitative analysis of the numbers of the immunoreactive puncta in each marker. vGlut1-and PSD95-expressing excitatory synapses were decreased, whereas VGAT-and gephyrin-expressing inhibitory synapses were increased in the TAC-treated hippocampal neurons, compared with those in the VH group. e, f Representative immunoblot and quantification of synaptic markers showed that TAC treatment significantly decreased the excitatory synaptic markers vGlut1 and PSD95 (e) and simultaneously increased the inhibitory synaptic markers VGAT and gephyrin (f). Optical densities of the bands in each lane were normalized with the β-actin optical density from the same gel. The values shown are presented as means ± SE (n = 5). *P < 0.05 vs. VH. Scale bar = 4 µm for a and b ◂ Fig. 3 Quantitative analysis for neurotransmitter receptors in the hippocampi of TAC-treated mice. The mRNA expression of neurotransmitter receptors was detected with RT-qPCR in the VH and TAC-treated hippocampi. Relative mRNA expression levels were calculated after normalization to GAPDH expression, and expression levels in VH were considered as the control. The values shown are presented as mean ± SE (n = 5). *P < 0.05 vs. control

Influence of TAC Treatment on Klotho Expression in the 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 found to be expressed in the pyramidal neurons in the hippocampal CA1 region, as assessed with 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 the 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 in 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. 4a), 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 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, e-h), whereas the TAC group showed weak Klotho immunoreactivity in the neurons (Fig. 4d, 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 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 Oxidative Stress in the Hippocampus
To investigate whether Klotho downregulation is associated with oxidative stress regulation after TAC treatment, we stained 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 Fig. 4 Decreased hippocampal expression of Klotho following chronic TAC treatment. The labeling intensity for Klotho was prominent in the vehicle (VH) groups, compared with the other groups (a). Representative immunoblot and quantification (b) of Klotho show that TAC treatment significantly decreased the level of Klotho in a dose-dependent manner. Optical densities of the bands in each lane were normalized with the β-actin optical density from the same gel. c, d Immunoreactivity for Klotho was detected in the pyramidal cell layer (pcl) and stratum radiatum (sr) of the CA1 region, and the labeling intensity was more prominent in the VH group (c) compared with that in the TAC group (d). e-l Triple labeling for Klotho with β-III tubulin and microtubule-associated protein 2 (MAP2), which are both cytoskeleton markers for neurons, indicated that Klotho expression is present in the soma and neurites of hippocampal neurons. e, i High-magnification images of the boxed areas in c and d, respectively. The punctate Klotho-labeled profiles were distributed in the stratum radiatum and are associated with the neurites of hippocampal neurons. Scale bar = 25 µm for c and d and 10 µm for e-l. The values shown are presented as means ± SE. *P < 0.05 vs. VH. Scale bar = 400 µm for a ◂ 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][24][25], we examined the increased oxidative stress in the hippocampi 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.

Discussion
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 [57][58][59][60][61][62]. 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 a synaptic imbalance in the hippocampus.
Klotho plays an important role in cognitive function in the hippocampus [33-35, 63, 64]. However, the localization of Klotho in the synapse remains unclear. Therefore, we evaluated the distribution of Klotho expression in the hippocampus. Examining the hippocampal CA1 region, identifying the phenotype of Klotho-expressing cells, and examining the subcellular localization of Klotho revealed that Klotho was expressed in neuronal processes and was localized to the postsynaptic neurites at the subcellular level. To our knowledge, this is the first report on the anatomical localization of Klotho expression in the synapse.
Next, the influence of TAC on the changes in Klotho was examined. TAC downregulated hippocampal Klotho in a dose-dependent manner. As previously reported [65], Klotho expression was localized in hippocampal neurons, and TAC treatment downregulated Klotho in neurons. Further, TAC treatment downregulated Klotho at excitatory, but Overall, these findings suggest that TAC causes a 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. Therefore, we propose that TAC-induced impairment of cognitive function is associated with synaptic imbalance via Klotho downregulation at hippocampal excitatory synapses.
Our previous study reported that Klotho is closely associated with aggravated TAC-induced oxidative stress in the kidney [37,66]. The present 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 [20,37,[67][68][69]. Klotho inhibits PI3K and Akt, activates FoxO3a, and increases MnSOD expression, thereby promoting ROS clearance and increasing the resistance to oxidative stress [39,70]. This suggests that Klotho may attenuate ROS-related oxidative stress. However, the role of Akt in neuronal oxidative injury remains obscure. Several mechanisms were demonstrated to be associated with oxidative stress in neurons. The cells get more susceptible to oxidative damage as oxidative stress increases due to the activation of Akt [71]. The increased Akt-dependent oxidative phosphorylation and oxygen consumption may result in persistence of the pro-oxidant state. In addition, overactivated Akt could consistently suppress the FoxO transcription factor, particularly FoxO3a, which increases the expression of antioxidant proteins such as SOD 2, catalase, and sestrins [72,73]. A previous study reported that FoxO3a-dependent decreased SOD2 expression rendered an experimental model more vulnerable to oxidative stress [74]. High-magnification views of the boxed areas in e. Klotho was expressed in the MAP2-positive neuronal dendrites; the arrowheads indicate the postsynaptic density (PSD) in the dendrites. g, h Pre-embedding immunoelectron microscopic images of Klotho immunostaining obtained using immunoperoxidase (g) and immunogold-silver labeling (h). Klotho-positive electron-dense (black asterisk in g) regions were associated with the postsynaptic profile along with the PSD (arrowheads in g) and were located adjacent to the presynaptic terminals with numerous synaptic vesicles (white asterisk in g). In addition, the silver grains for Klotho (arrows in h) were observed in the dendritic cytoplasm with PSD (arrowheads in h). The white asterisks indicate synaptic vesicles in the presynaptic profiles. Scale bar = 3 µm for a-d, 1 µm for e, 0.5 µm for f, and 200 nm for g and h Our results indicate that TAC increased the levels of 8-OHdG, an oxidative damage marker; activated the PI3K/ AKT-mediated phosphorylation of FoxO3a; and inhibited FoxO3a binding to the MnSOD promoter, thereby leading to reduced MnSOD expression (Fig. 7i, j). Thus, TAC-induced oxidative stress downregulated Klotho via PI3K/AKT pathway activation in the hippocampus.
Several mechanisms were reported to possibly be involved in TAC-induced neurotoxicity. In the central nervous system (CNS), calcineurin is highly expressed in neurons vulnerable to ischemic and traumatic injury. The CNIs, such as TAC, can cause selective toxic effects due to damage to neurons, glial cells, and oligodendrocytes with high calcineurin content [75,76]. In addition, CNIs can increase oxidative stress by directly altering mitochondrial function [77]. Moreover, CNIs can alter the excitability properties of neurons by modulating the activity of excitatory and inhibitory neurotransmitter receptors [78,79], thereby leading to membrane depolarization [80].
CNI treatment is associated with neurotoxicity [81]. However, the influence of CNIs on cognitive function remains controversial [82][83][84][85][86], possibly owing to the diverse etiology of cognitive impairment after organ transplantation, including age and underlying diseases. Our study clearly defines the association between CNI and cognitive function. Impaired cognitive function in organ transplant recipients results in the lowering of the quality of life (including problems with 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 longterm 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 Klotho-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 a synaptic imbalance in the hippocampus, and that these processes may be associated with Klotho downregulation through TAC-induced oxidative stress (Fig. 9).