Regulation of Life Extension Factor Klotho on Depressive-like Behaviors via Modulation of GluN2B Function in the Nucleus Accumbens

Klotho is a life extension factor that has an ability to regulate the function of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs), whose dysfunction in the nucleus accumbens (NAc) underlies critical aspects of the pathophysiology of major depression. Here we study the functional relevance of klotho in the pathogenesis of depression. A chronic social defeat stress paradigm, where mice are either categorized as susceptible or unsusceptible group based on their performance in a social interaction test, was used in this study. We found that the expression of klotho was largely decreased in the NAc of susceptible mice when compared to control or unsusceptible group. Genetic knockdown of klotho in the NAc induced depressive-like behaviors in naive mice, while overexpression of klotho produced an antidepressive effect in normal mice and ameliorated the depressive-like behaviors in susceptible mice. Molecularly, knockdown of klotho in the NAc resulted in selective decreases of total and synaptic GluN2B expression that were identical to susceptible mice. Elevation of klotho in the NAc reversed the reductions of GluN2B expressions, as well as altered synaptic transmission and spine density in the NAc of susceptible mice. Furthermore, blockade of GluN2B with a specic antagonist abolished the benecial effects of klotho elevation in susceptible mice. Collectively, we demonstrated that klotho in the NAc modulates depressive-like behaviors by regulating the function of GluN2B-containing NMDARs. These results reveal a novel role for klotho in the pathogenesis of depression, opening new insights into the molecular basis of major depression. these results demonstrated that klotho in the NAc could modulate depressive-like Previous studies have proved that synaptic molecular adaptations occurring in the neurons of NAc underlie susceptible and resilient responses to chronic stress 8 . We next performed a set of electrophysiological experiments to investigate the modulatory effects of klotho on synaptic plasticity at cortico-accumbal glutamatergic synapses. We rstly conducted whole-cell voltage-clamp recordings of synaptically evoked NMDAR-mediated excitatory postsynaptic currents (EPSCs) in NAc slices to examine the modulation of GluN2B-NMDARs. The relative contribution of GluN2B to EPSCs was determined by measuring the sensitivity of EPSCs to Ro 25-6981, a second-generation NMDAR blocker that displays a 3000-fold higher specicity to the GluN2B subunit than to other subunits. Compared to control group, Ro 25-6981-sensitive EPSCs were substantially reduced in the NAc neurons of susceptible mice (control: 34.5 ± 5.9% of baseline, susceptible: 5.8 ± 3.1% of baseline; p < 0.01), and overexpression of klotho in the NAc signicantly increased Ro 25-6981-sensitive EPSCs in susceptible mice to a level comparable to control (31.7 ± 5.9% of baseline, p < 0.01 vs. susceptible group) (Figure 5a and b). These results indicate that elevation of klotho could reverse the detrimental effect of CSDS on GluN2B-mediated function. The PPF, a sensitive measure of the probability of transmitter release, is a common form of short-term presynaptic plasticity. As shown in Figure S5a and b, CSDS did not affect the PPF at cortico-accumbal pathway, and klotho overexpression in susceptible mice also had no effect on PPF, suggesting the lack of gross change in presynaptic function. Then the


Introduction
Depression is a common type of affective disorders that is characterized by marked and persistent low mood and loss of interest, leading to serious functional impairments in patients 1 . The Global Burden of Disease Study demonstrates that depression is the second leading cause of disability-adjusted life-years in 2020 2 . Numerous antidepressants are currently used in clinical practice, however, it still takes weeks for patients to get therapeutic effects and some patients are poor tolerant to antidepressants 3 .
Understanding of the pathophysiological mechanisms underlying major depression would shed light on the treatment of major depressive disorders.
The nucleus accumbens (NAc), a brain region located in the ventral aspect of the basal ganglia, is widely recognized as the center of reward and motivation 4 . NAc medium spiny neurons display a two-state membrane potential controlled by active channels and synaptic input 5 , thereby affecting the functional activity in networks that underlie cognition and behavior 6 . The NAc receives glutamatergic inputs from the medial prefrontal cortex (mPFC), basolateral amygdala and hippocampus. Accumulating evidence has shown that altered glutamatergic transmission in the NAc contributes importantly to the pathophysiology of depression [7][8][9][10] . Synaptic glutamate release in mPFC-NAc is decreased in mice exhibiting depressive-like behaviors 9 , and the decreased activity at glutamatergic synapses obstructs the later induction of long-term depression (LTD) in the NAc of these mice 11,12 . Our previous study revealed that chronic stress caused persistent downregulation of total and synaptic GluN2B, a N-methyl-D-aspartate receptor (NMDAR) subunit with key functions in learning and memory, in the NAc and disrupted the induction of NMDAR-dependent LTD of cortico-accumbal glutamatergic synapse, while restoration of GluN2B loss reversed stress-induced LTD de cit and alleviate depressive-like behaviors in chronic social defeat stress (CSDS)-susceptible mice, indicating that downregulation of GluN2B function in the NAc underlies the synaptic and behavioral adaptations to chronic stress 8 .
The klotho protein is a recently discovered protein that is associated with life extension. Overexpression of klotho extends life span, whereas loss of klotho leads to an accelerated aging and short life [13][14][15] . In addition to lifespan extension, it has also been linked to cognition and other neuropsychiatric disorders.
For example, knockout of klotho resulted in memory retention de cits in mice 16 , while elevation of klotho expression can enhance hippocampus-dependent learning and memory in normal rodents and protect against cognitive decline in animal models of Alzheimer's disease (AD) [17][18][19] . In a previous clinical observational study, AA Prather 20 found that women under high chronic stress displayed signi cantly lower levels of serum klotho compared with low-stress controls. KLOTH gene variants in uenced the response to selective serotonin reuptake inhibitors (SSRIs) in late-life major depressive disorder 21 . Electroconvulsive therapy (ECT), a highly effective antidepressant treatment, signi cantly enhanced the levels of klotho in the cerebrospinal uid of geriatric patients with major depression 22 . However, whether klotho is involved in the pathogenesis of major depression remains unclear.
Klotho is highly expressed in the choroid plexus and neurons, as well as in the kidney and reproductive organs. Its transmembrane form can be released by sheddases and circulate in serum and cerebrospinal uid throughout life 23 . There are studies demonstrating that klotho could enhance the function of GluN2B subunit 18, 19 . Upregulation of klotho expression promoted hippocampal synaptic plasticity and cognition by enriching synaptic GluN2B in the hippocampus of mice, while blocking GluN2B abolished the bene cial effects of klotho elevation 18 . Elevating klotho expression in human amyloid precursor protein (hAPP) transgenic mice increased the abundance of GluN2B in the postsynaptic densities to improve spatial learning and memory 24 . In view of the important role of accumbal GluN2B loss in the pathophysiology of depression 8 , as well as the regulation of klotho on GluN2B function, it is possible that klotho in the NAc also ful lls important functions in the pathogenesis of major depression. To test this hypothesis, we rst investigated whether the expression of klotho was changed in the NAc from mice displaying depressive-like behaviors and then explored the in uences of modulating accumbal klotho expression on depressive-like behaviors in mice. By constructing stable adeno-associated virus vector system to regulate the expression of klotho in the NAc of mice, we demonstrated that klotho in the NAc modulated depressive-like behaviors in mice by regulating the surface stability of GluN2B subunit.

Animals
Adult male C57BL/6J mice (8-10 weeks old) and male CD1 mice (6-month-old) were purchased from Hunan SJA Laboratory Animal Co., Ltd (Changsha, Hunan, China). Mice were fed under standard conditions (12 h light/dark cycle; lights on from 07:00 to 19:00; 23 ± 1°C ambient temperature; 55 ± 10% relative humidity), with free access to food and water. All behavioral experiments were performed in the day and conducted in compliance with the Guide for the Care and Use of Laboratory Animals (8th edition, Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences, Washington DC). This research was approved by the Review Committee for the Use of Human or Animal Subjects of Jiangxi Mental Hospital. Experimenters were blinded to experimental group and the order of testing was counterbalanced during behavioral experiments.

Social Interaction Test (SIT)
Social avoidance behavior was assessed with a novel CD1 mice in a two-stage social interaction test. In the rst 3-min test (target absent), the defeated mice were allowed to freely explore an arena (44 × 44 cm) containing a plexiglass and wire mesh enclosure (10 × 6 cm) against one wall of the arena. In the second 3-min test (target present), the experimental mice were returned to the area with a novel CD1 mice enclosed in the plexiglass wire mesh cage. This allowed the animal to see, hear, and smell, but not physically contact. Time spent in the 'interaction zone' (14 × 26 cm) surrounding the plexiglass wire mesh cage and 'distance travelled' within the arena was recorded by ANY-maze tracking software (ANY-maze, Wood Dale, IL). The segregation of susceptible and unsusceptible mice was based on the social interaction ratio, which was calculated as: time in interaction zone when target present/time in interaction zone when target absent × 100%. Defeated mice with a social interaction ratio < 100% were de ned as 'susceptible', while those with a social interaction ratio ≥ 100% were de ned as 'unsusceptible'.
Additional experimental procedures and statistics are described in Supplement information.

CSDS Signi cantly Decreased Klotho Expression in the NAc of Susceptible Mice
The CSDS has a good predictive validity for modeling the symptomatology of depression, thus we adopted this model to investigate the role of klotho in depression in this study. C57BL/6 J mice were exposed to 10 consecutive days of stress and then were designated as susceptible or unsusceptible mice based on social interaction ratios ( Figure S1a). Compared to control and unsusceptible mice, susceptible mice displayed a signi cant decrease in the sucrose preference ( Figure S1b To explore whether CSDS would result in change of klotho expression in the brain, the protein level of klotho was determined by western blot analysis. As shown in Figure 1a, klotho protein expression in the whole-brain was signi cantly decreased in susceptible mice compared with control or unsusceptible mice (p < 0.01). Then the expressions of klotho in brain regions closely related to depression, including PFC, hippocampus and NAc, were detected. As shown in Figure 1b, there was no signi cant difference in klotho expression in the PFC among control, unsusceptible and susceptible group. However, compared to control or unsusceptible mice, the levels of klotho in the hippocampus and NAc of susceptible mice were signi cantly decreased. Klotho proteins in the hippocampus and NAc of susceptible mice were reduced by 25.2 ± 3.7% (p < 0.05 vs. control) and 63.2 ± 5.2% (p < 0.01 vs. control), respectively.

Genetic Knockdown of Klotho in the NAc Induced Depressive-like Behaviors in Mice
Given that the level of klotho protein was largely decreased in the NAc of mice after CSDS, we presumed that abnormal accumbal klotho signaling might contribute to the pathogenesis of depression. To test this hypothesis, we investigated whether downregulation of klotho expression in the NAc would result in depressive-like behaviors in mice ( Figure 1c). We used an adeno-associated viral vector (AAV) to speci cally reduce the expression of klotho in the NAc. AAV-klotho knockdown-GFP (KL-KD) or AAV-Null-GFP (GFP) was stereotaxically infused into the NAc of naive mice. The expression of klotho in the NAc was detected 2 weeks after injection. We showed that numerous GFP-positive cells were found in the NAc region after injection ( Figure 1d) and the protein expression of klotho in the NAc of KL-KD mice was signi cantly decreased when compared to control or GFP group (Figure 1e, p < 0.01 vs. control), indicating that AAV-mediated knockdown of klotho in the NAc was successfully constructed.
Next, we examined whether mice with knockdown of klotho in the NAc displayed depressive-like behaviors. As expect, compared to control mice, the KL-KD mice had a signi cantly lower level of sucrose consumption ( Figure 1f, p < 0.01 vs. control group). The immobility time of mice in the KL-KD group was also signi cantly increased in FST and TST (Figure 1g and h, p < 0.01 vs. control group). Open eld test showed that there was no signi cant difference in traveled distance among groups (p > 0.05), while the time spent in the center square was signi cantly decreased in KL-KD mice (Figure 1i and j, p < 0.01 vs. control group), indicating that the behavioral alterations in KL-KD mice were not due to change in spontaneous locomotor activity, but rather depressive-like behaviors. AAV-control-GFP (GFP) mice did not display any behavioral difference from the control group. The results indicated that reduced nucleus accumbens klotho contributed to the pathogenesis of major depression.

Genetic Overexpression of Klotho in the NAc Produces Antidepressive Effects in Both Normal and CSDS Susceptible Mice
To verify the role of klotho signaling in depression, we investigated whether elevation of klotho expression in the NAc would affect depressive-like behaviors. We rst tested the in uence of elevating accumbal klotho on depressive-like behaviors in normal mice. AAV was employed to enhance klotho expression in the NAc. AAV-klotho overexpression-GFP (KL-OE) or AAV-Null-GFP (GFP) was stereotaxically infused into the NAc of normal mice. Two weeks after injection, the protein expression of klotho in the NAc of KL-OE mice was signi cantly increased when compared to control or GFP group ( Figure S2a, p < 0.01 vs. control or GFP group). Increased sucrose consumption in SPT and decreased immobility time in FST were observed in these KL-OE mice ( Figure S2b and c, both p < 0.05 vs. control group), indicating that elevation of accumbal klotho could exhibit an antidepressive effect in normal mice.
Then we further explored whether elevation of klotho expression in the NAc would attenuate the depressive-like behaviors in CSDS susceptible mice ( Figure 2a). As shown in Figure 2b, KL-OE increased the protein expression of klotho in the NAc of susceptible mice to a level comparable to that of control group (p < 0.01 vs. susceptible or susceptible-GFP group). Results from social interaction test revealed that accumbal KL-OE signi cantly increased the exploration time in interaction zone and the interaction ratio in susceptible mice (Figure 2c To explore the potential mechanisms underlying the modulatory effects of klotho on depressive-like behaviors, we turned our attention to NMDARs, whose dysfunction were demonstrated to mediate behavioral and synaptic adaptations to chronic stress 8 . We performed western blotting to examine the levels of NMDAR subunits and postsynaptic density protein 95 (PSD-95) in total protein homogenates of NAc 10 days after CSDS. As previous reported 8 , we showed that total protein expressions of GluN1 and GluN2A in the NAc were not changed after CSDS (p > 0.05 vs. control), while the level of GluN2B subunit in the NAc of susceptible mice were signi cantly decreased compared to control group ( Figure S3a, p < 0.01). PSD-95 is a neuronal PDZ protein that associates with NMDARs at synapses to facilitate downstream intracellular signaling and modulate synaptic plasticity 25,26 . Akin to GluN2B, PSD-95 displayed a signi cant decrease in the NAc of susceptible mice ( Figure S3a, p < 0.01 vs. control). Then we investigated whether downregulation of klotho in the NAc would result in similar changes in NMDAR subunit expression. As shown in Figure 3a, KL-KD in the NAc caused a speci c decrease in total protein levels of GluN2B subunit and PSD-95, but did not alter total protein levels of GluN1 and GluN2A (p < 0.01 vs. control or GFP group).
The biological consequences of NMDAR activation mainly depend on whether the receptors are located in synaptic or extrasynaptic sites 27 . We therefore detected the surface expressions of NMDAR subunits in NAc of mice using a protein cross-linking assay that speci cally detects synaptic proteins. Similar to previous report 8 , we showed that there was no difference in the levels of GluN1 and GluN2A in both surface pool and intracellular pool between control and susceptible mice ( Figure S3b  To further con rm the action target of klotho on depressive-like behaviors, we explored the in uences of elevating accumbal klotho on GluN2B expression in NAc of mice. We rst detected the total and surface expressions of GluN2B in NAc in mice with klotho overexpression. As shown in Figure S4a, genetic overexpression of klotho in the NAc in normal mice did not affect the total expressions of GluN1 and GluN2A subunit, but signi cantly increased the levels of total GluN2B and PSD-95 (p < 0.01 vs. control group). Protein cross-linking assay revealed that the surface expression of GluN2B in NAc was signi cantly increased by klotho expression ( Figure S4b, p < 0.01 vs. susceptible group).
We next investigated whether elevation of klotho could restore total and surface expressions of GluN2B in NAc of susceptible mice. As shown in Figure 4a, genetic overexpression of klotho in the NAc in susceptible mice had no effect on the expression levels of GluN1 and GluN2A subunit, but signi cantly increased the total expressions of GluN2B and PSD-95 to levels comparable to those in control group (p < 0.01 vs. susceptible group). Results from the BS 3 cross-linking experiments showed that genetic overexpression of klotho in the NAc in susceptible mice had no effect on the levels of GluN1 and GluN2A subunit in both the surface and intracellular pool (Figure 4b and c), but signi cantly increased the surface expression of GluN2B in NAc (Figure 4d, p < 0.01 vs. susceptible group). Together with the above data, these results demonstrated that klotho in the NAc could modulate depressive-like behaviors by regulating the stability of surface GluN2B.

Genetic Overexpression of Accumbal Klotho Reversed Altered Synaptic and Structural Plasticity in CSDS Susceptible Mice
Previous studies have proved that synaptic molecular adaptations occurring in the neurons of NAc underlie susceptible and resilient responses to chronic stress 8 . We next performed a set of electrophysiological experiments to investigate the modulatory effects of klotho on synaptic plasticity at cortico-accumbal glutamatergic synapses. We rstly conducted whole-cell voltage-clamp recordings of synaptically evoked NMDAR-mediated excitatory postsynaptic currents (EPSCs) in NAc slices to examine the modulation of GluN2B-NMDARs. The relative contribution of GluN2B to EPSCs was determined by measuring the sensitivity of EPSCs to Ro 25-6981, a second-generation NMDAR blocker that displays a 3000-fold higher speci city to the GluN2B subunit than to other subunits. Compared to control group, Ro 25-6981-sensitive EPSCs were substantially reduced in the NAc neurons of susceptible mice (control: 34.5 ± 5.9% of baseline, susceptible: 5.8 ± 3.1% of baseline; p < 0.01), and overexpression of klotho in the NAc signi cantly increased Ro 25-6981-sensitive EPSCs in susceptible mice to a level comparable to control (31.7 ± 5.9% of baseline, p < 0.01 vs. susceptible group) (Figure 5a and b). These results indicate that elevation of klotho could reverse the detrimental effect of CSDS on GluN2B-mediated function.
The PPF, a sensitive measure of the probability of transmitter release, is a common form of short-term presynaptic plasticity. As shown in Figure S5a and b, CSDS did not affect the PPF at cortico-accumbal pathway, and klotho overexpression in susceptible mice also had no effect on PPF, suggesting the lack of gross change in presynaptic function. Then the input-output relationships for eld excitatory postsynaptic potentials amplitude, an indicator of synaptic e cacy, were compared among groups. We found that there was a slight decrease in the amplitude of eld excitatory postsynaptic potentials in the corticoaccumbal pathway in susceptible mice, while genetic overexpression of accumbal klotho reversed the decreased basal synapse transmission in these mice ( Figure S5c and d, p < 0.05 vs. susceptible group).
Persistent impairment in NMDAR-dependent LTD in NAc was associated with behavioral adaptations to chronic stress 8 . Thus, the NMDAR-LTD in the NAc was compared in the control, susceptible, and KL-OE susceptible groups. Consistent with previous study, the NMDAR-LTD was disrupted in susceptible mice (Figure 5d and g, p < 0.05 vs. control group). Genetic overexpression of accumbal klotho signi cantly reversed the disrupted NMDAR-LTD in NAc of susceptible mice (Figure 5f and g, p < 0.05 vs. susceptible and GFP group). This result indicated that regulation of accumbal klotho could normalize the impaired synaptic plasticity that was associated with depressive behaviors.
To further characterize the mechanisms underlying the modulation of klotho in depressive-like behaviors, we investigated the effects of klotho elevation on structural plasticity in susceptible mice. Golgi staining was employed to determine dendrite spine density in neurons of NAc. Consistent with previous reported 28 , the dendrite spine density in neurons of NAc was signi cantly increased in susceptible mice (Figure 5j and k, p < 0.05 vs. control group). Genetic overexpression of accumbal klotho in susceptible mice signi cantly reversed the alteration of dendrite spine density in NAc (Figure 5j and k, p < 0.05 vs. susceptible mice), demonstrating that elevation of accumbal klotho normalized structural plasticity in susceptible mice.

Blockade of GluN2B Abolished the Bene cial Effects of Klotho Elevation in CSDS Susceptible Mice
We then investigated whether blocking GluN2B-containing NMDARs would eliminate the bene cial effects of klotho elevation in susceptible mice. Ro 25-6981 is usually used in the 0.1-1 µM range in brain in vitro 29 . Given that blockade of GluN2B may have impacts on synaptic plasticity and depressive-like behaviors, low dose of Ro 25-6981 was used in this study to avoid this possibility as far as possible. We showed that bilaterally intra-NAc infusion of low dose of Ro 25-6981 (0.1 µM, 0.5 µl) at 20 min before social interaction or sucrose preference test had no signi cant effect on the exploration time in interaction zone and the interaction ratio, as well as sucrose preference in susceptible mice, while it signi cantly abolished the increased social interaction and sucrose preference induced by klotho overexpression in these mice (Figure 6a and b, p < 0.01 vs. KL-OE susceptible mice). Post-hoc comparisons using Bonferroni's test showed that both the interaction ratio and the sucrose consumption in klotho-overexpressed susceptible mice that were also treated with Ro 25-6981 were not different from those in GFP-treated susceptible mice (p > 0.05). In a separate set of experiments, we investigated the in uence of an acute administration of Ro 25-6981 on NMDAR-dependent LTD in NAc of mice. Similar to the behavioral results, bath application of Ro 25-6981 (0.1 µM) for 20 min did not affect LFS-induced LTD in slices from susceptible mice (Figure 6e), but signi cantly eliminated the bene t of klotho overexpression on LTD in these mice (p < 0.01 vs. KL-OE susceptible mice; Figure 6g). Post-hoc comparisons showed that the level of LTD in klotho-overexpressed susceptible mice with bath application of Ro 25-6981 was comparable to that of susceptible mice (p > 0.05). These data indicate that upregulation of GluN2B-NMDAR function mediated the bene cial effects of klotho elevation in susceptible mice.

Discussion
In the present study, we demonstrated a critical role of accumbal klotho in the pathogenesis of major depression. Exposure to chronic stress led to a signi cant downregulation of klotho in the NAc of mice and genetic knockdown of klotho in the NAc induced depressive-like behaviors. Overexpression of klotho in the NAc produced an antidepressive effect in normal mice, and ameliorated depressive-like performances and reversed the alterations of synaptic plasticity and structural morphology in CSDS susceptible mice. The molecular effects of klotho might be correlated with the regulation of GluN2Bcontaining NMDAR function because klotho knockdown in the NAc resulted in selective decreases of total and synaptic GluN2B expression, which were identical to those observed in susceptible mice, and elevation of accumbal klotho could reverse the changes of GluN2B expression. Moreover, a GluN2B-speci c antagonist abolished the bene ts of klotho elevation on depressive-like performances and accumbal LTD in susceptible mice. These ndings demonstrate that klotho in the NAc modulates depressive-like behaviors by regulating the function of GluN2B-containing NMDARs.
Klotho is a single-pass membrane-bound protein that can be alternatively spliced to a membrane bound form (m-KL) and secreted form (s-KL) 30 . It is released and cleaved in cerebrospinal uid (CSF) and plasma, and has an in uence on longevity and susceptibility to multiple complex disorders, including atherosclerosis, stroke and depression 31 . Klotho mutant mice display an increased level of oxidative stress in the hippocampus at 5 weeks of age and impaired cognitive function at 7 weeks 16 . Women under high chronic stress had signi cantly lower levels of serum klotho when compared to low-stress controls 20 . The levels of klotho in the CSF were also signi cantly increased in geriatric patients with severe depression after electroconvulsive therapy, a highly effective antidepressant treatment strategy 22 . In this study, we found that chronic stress resulted in a signi cant decrease in klotho expression in the NAc. Previous studies have demonstrated that blunted responses in the NAc to gain were observed in depressed individuals 32 as well as the offspring and rst-degree relatives of depressed individuals 33 .
Rappaport et al. reported that current depression severity was associated with hyporeactivity in the NAc in response to the anticipation of a reward 34 . We showed that genetic knockdown of klotho in the NAc induced depressive-like behaviors in mice, and genetic overexpression of accumbal klotho obviously ameliorated depressive-like behaviors in susceptible mice. These data indicate that reduced nucleus accumbens klotho contributes to the pathogenesis of major depression.
Synaptic plasticity is the activity-dependent modi cation of the strength or e cacy of synaptic transmission at synapses and has been demonstrated to play a central role in the capacity of the brain to incorporate transient experiences into persistent memory traces 35 . It has been proposed that activitydependent remodeling of excitatory synapses and associated dendritic spines is impaired during chronic stress, leading to neurological circuit disorders in the brain and the onset of depression symptoms 36 . NAc neurons receive glutamatergic inputs arising from limbic and cortical regions. Stress-induced dysfunction in the synaptic plasticity of cortico-accumbal glutamatergic synapse is implicated in the symptomology of depression 37 . Consistent with previous report 8 , we showed that there was a signi cant decrease in excitatory postsynaptic responses and an impairment of NMDAR-dependent LTD in the NAc of susceptible mice. Overexpression of klotho in the NAc signi cantly reversed the reduced postsynaptic responses and impaired LTD in susceptible mice, indicating that klotho elevation could restore the synaptic function in the NAc of susceptible mice. Alterations in synaptic strength or connectivity of neurons are responsible for the long-lasting behavioral symptoms induced by chronic stress 28, 38-40 . After chronic social defeat stress, medium spiny neurons (MSNs) of NAc exhibit increased spine density that is correlated with enhanced depressive behaviors 40,41 . We found that the dendrite spine density in the MSNs of NAc was signi cantly increased in susceptible mice, while genetic overexpression of accumbal klotho substantially reversed the alteration of dendrite spine density in these mice, demonstrating elevation of accumbal klotho could normalize the structural plasticity in depressed mice. These results that klotho elevation reversed the disruptions of synaptic and structural plasticity in susceptible mice provided supporting evidence for the bene t of elevating klotho on depressive-like behaviors.
Previous data from our study showed that the reduction of GluN2B in the NAc can aggravate depressivelike behavior in mice. It is worth noting that systemic administration of NMDA receptor antagonist, such as Ro 25-6981 and ketamine, is capable of exerting signi cant antidepressant effects in both model animals and depressive patients 42 . This fact seems to con ict with our nding. However, there are some possible reasons to explain this discrepancy. Firstly, the mechanisms by which ketamine exert antidepressant effects are complex. Recent studies suggest that the effects of ketamine on depression cover not only NMDAR antagonism, but also include glutamate surge, reduced inhibitory GABAergic transmission, AMPAR-mediated increase in mTOR-dependent neuroplasticity as well as BDNF release [43][44][45][46][47] . On the other hand, different brain regions might exert different effects due to different structural components. A major proportion of NAc neurons are MSNs 48 . During the course of depression, NAc region would undergo some changes in protein expressions and functional connectivity that are different from other brain regions. For example, chronic stress causes a reduction in hippocampal BDNF expression and spine density 49,50 , while increased BDNF expression and dendrite spine density were observed in the NAc after chronic stress 51 . Our previous study has also demonstrated that chronic stress resulted in a long-lasting reduction of GluN2B in the NAc, which could be restored by uoxetine treatment, and unsusceptible mice showed patterns of GluN2B regulation that overlapped dramatically with those seen with uoxetine treatment 8 . In this study, we showed that chronic stress caused a parallel change in GluN2B and klotho expression in the NAc and the total and surface expressions of GluN2B in the NAc can be regulated by altering klotho levels via AAV-mediated knockdown or overexpression. Furthermore, treatment with low dose of Ro 25-6981 had no signi cant effect on the depressive-like behaviors and NMDAR-LTD in susceptible mice, but signi cantly abolished the bene cial effects of klotho overexpression in these mice, indicating that GluN2B was the action target of klotho in modulation of depressive-like behaviors. However, how klotho regulates the levels of total GluN2B protein and enriches GluN2B within synapses, directly or indirectly, remains to be determined but may implicate regulations of translation, posttranslational modi cation, recycling, or tra cking of the subunit. It also remains to be determined whether the effects of klotho elevation on GluN2B are mediated by the transmembrane or secreted form of klotho.
Taken together, we preliminarily demonstrated that klotho in the NAc modulate depressive-like behaviors by regulating the function of GluN2B-containing NMDARs. This nding provides novel insights into the pathogenesis of major depression and regulation of klotho in the NAc might be a strategy for the treatment of depression. However, how chronic stress causes a change in the expression of klotho in the NAc still needs further study.

Data availability
The authors con rm that the data supporting the ndings of this study are available within the article and the Supplementary Material. Additional data related to this paper may be requested from the authors.  klotho produced no effect on the expressions of GluN1 (b) and GluN2A (c) in both the surface and intracellular pools in NAc of mice. (d) Genetic knockdown of accumbal klotho signi cantly reduced GluN2B expression in the surface pool but not in the intracellular pool (n = 4 mice per group). All data were presented as normalized mean ± SEM. ## p < 0.01 vs. GFP group. Statistical results show that CSDS caused a reduction of GluN2B in the surface pool of NAc, while klotho overexpression reversed the decline of surface GluN2B expression (n = 6 mice per group). All data were presented as normalized mean ± SEM. ** p < 0.01 vs. control; ## p < 0.01 vs. susceptible group.  display the median, rst and third quartiles (boxes), and the min-max (whiskers), and other data were presented as normalized mean ± SEM. ** p < 0.01 vs. control; ## p < 0.01 vs. Sus + GFP group; && p < 0.01 vs. Sus + KL-OE group.

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