CSDS Significantly 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 significant decrease in the sucrose preference (Figure S1b, p < 0.01) and a significant increase in immobility time in both the FST and the TST (Figure S1c and d, p < 0.01). These behavioral results demonstrated that CSDS could successfully induce depressive-like behaviors in mice. Therefore, CSDS susceptible mice were used as the model mice of major depression in the subsequent experiments.
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 significantly 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 significant 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 significantly 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 specifically 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 significantly 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 significantly 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 significantly increased in FST and TST (Figure 1g and h, p < 0.01 vs. control group). Open field test showed that there was no significant difference in traveled distance among groups (p > 0.05), while the time spent in the center square was significantly 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 first tested the influence 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 significantly 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 significantly increased the exploration time in interaction zone and the interaction ratio in susceptible mice (Figure 2c, p < 0.01 vs. susceptible or susceptible-GFP group). The decreased sucrose preference in susceptible mice was also significantly reversed by KL-OE (Figure 2d, p < 0.01 vs. susceptible or susceptible-GFP group). Furthermore, accumbal KL-OE in susceptible mice shorten the immobility time in FST (Figure 2e) and TST (Figure 2f) to levels comparable to those of control group (p < 0.01 vs. susceptible or susceptible-GFP group). Open field test showed that accumbal KL-OE in susceptible mice did not affect the traveled distance of mice, but increased the time spent in the center (Figure 2g, p < 0.01 vs. susceptible or susceptible-GFP group). These results demonstrated that elevation of klotho in the NAc ameliorated depressive-like behaviors of susceptible mice, confirming the critical modulatory effects of accumbal klotho on the pathogenesis of major depression.
Knockdown of Klotho in the NAc Resulted in Selective Decreases of Total and Synaptic GluN2B that Were Identical to CSDS Susceptible Mice
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 stress8. 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 reported8, 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 significantly 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 plasticity25, 26. Akin to GluN2B, PSD-95 displayed a significant 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 specific 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 sites27. We therefore detected the surface expressions of NMDAR subunits in NAc of mice using a protein cross-linking assay that specifically detects synaptic proteins. Similar to previous report8, 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 and c, p > 0.05). However, a robust decrease in GluN2B was observed in the surface pool in susceptible mice (Figure S3d, p < 0.01). These results demonstrated a selective GluN2B downregulation in a specific subcellular compartment (surface membranes) in the NAc of depressive model mice. We next explored the influence of klotho downregulation on the expressions of NMDARs in the synapses of NAc. Similarly, there was no difference in the levels of GluN1 and GluN2A in both surface and intracellular pool between control and KL-KD mice (Figure 3b and c), while a significant decrease was observed in GluN2B in the surface pool in KL-KD mice (Figure 3d, p < 0.01 vs. control or GFP group). These results demonstrated that knockdown of klotho produced an identical change in GluN2B expression to susceptible mice, suggesting that decrease of accumbal klotho resulted in depressive-like behaviors in mice via a selective downregulation of GluN2B at synapses of NAc.
Genetic Overexpression of Klotho in the NAc Upregulated the Expressions of Accumbal GluN2B in Both Normal and CSDS Susceptible Mice
To further confirm the action target of klotho on depressive-like behaviors, we explored the influences of elevating accumbal klotho on GluN2B expression in NAc of mice. We first 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 significantly 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 significantly 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 significantly 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 BS3 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 significantly 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 stress8. We next performed a set of electrophysiological experiments to investigate the modulatory effects of klotho on synaptic plasticity at cortico-accumbal glutamatergic synapses. We firstly 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 specificity 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 significantly 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 field excitatory postsynaptic potentials amplitude, an indicator of synaptic efficacy, were compared among groups. We found that there was a slight decrease in the amplitude of field excitatory postsynaptic potentials in the cortico-accumbal 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 stress8. 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 significantly 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 reported28, the dendrite spine density in neurons of NAc was significantly increased in susceptible mice (Figure 5j and k, p < 0.05 vs. control group). Genetic overexpression of accumbal klotho in susceptible mice significantly 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.