p53 Knock Out Mice Enhances Anxiety- and Depression-Like Behaviors through an Increase of Glutamate, Calcium, and Cytokine-Mediated Cell Death Signals

Extensive epidemiological evidence indicates that patients with certain cancers have a lower probability of developing some types of neurodegenerative diseases (ND) and mood disorders and vice versa. These inverse comorbidities may be associated with several different molecular processes. p53, is a potentially responsible for regulating the development of ND, mood disorders as well as cancers. To investigate whether the tumor suppressor p53 may be associated with ND development, we studied the behavioral changes in p53 knockout (p53 −/− ) mice and possible action mechanisms. Increased anxiety-like but not depression-like behaviors were displayed in p53 −/− mice without impaired motor activities under the non-chronic unpredictable mild stress condition. However, in the p53 −/− mice, more anxiety-like and depression-like behaviors were observed in the chronic unpredictable mild stress (CUMS) condition. Our mechanism studies showed that brain-derived neurotrophic factor (BDNF) protein was signicantly downregulated, but glutamate levels were signicantly increased in the prefrontal cortex of p53 −/− mice. Further analyses showed that the p53 −/− mice caused more stress-induced nerve damage as a result of an increase in intracellular calcium levels and N-methyl D-aspartate receptor subtype 2B (NMDAR2B) expression. Treatment with corticosterone (mimics CUMS in vitro) increased glutamate levels, NMDAR2B expression, and calcium levels, and these levels were elevated by co-treatment with pithrin-α (p53 inhibitor) in PC12 cells. Cell death and cell death-mediated signals (p-p38, p-JNK and caspase-3) were upregulated, but neuroprotective signals (BDNF p-Akt, p-ERK and p-CREB) were downregulated in p53 −/− mice, and corticoid and/or pithrin-α treated PC12 cells. These data indicate that p53 may be an important preventive factor against depression and anxiety, and thus suggests a possible correlation between cancer and anxiety/depression development.


Introduction
Extensive epidemiological evidence indicates that patients with certain cancers have a lower probability of developing some types of neurodegenerative diseases (NDs) such as Alzheimer's disease (AD), Parkinson's disease (PD) and schizophrenia (SCZ), and vice versa 1 . These inverse comorbidities may be associated with several different molecular processes. A transcriptomic meta-analysis of three ND types and three cancer types (lung, prostate, and colorectal) demonstrated the inverse comorbidities, and signi cant overlapping genes were found to be upregulated in NDs but downregulated in cancers. They were also found to be downregulated in NDs but upregulated in cancers [2][3][4][5] . One of these molecules, p53, is potentially responsible for regulating the development of NDs and cancer [6][7][8] .
Several studies have also demonstrated that p53 could be involved in complex molecular interactions putatively associated with the inverse correlation between cancer and AD 6,9 . Moreover, p53 at the transcriptional level was recently shown to upregulate parkin, a gene responsible for the development of PD 10 . The upregulation of parkin levels could, in turn, contribute to the activation of p53 at the transcriptional level, which could explain, at least in part, the increased cellular apoptotic commitment in cerebral cancer 11 . Previous studies have demonstrated that the inhibition of p53 could be an important indicator for the treatment of neuropathic symptoms 12,13 .
Although many studies have supported the correlation among p53, tumor suppressor molecules, and ND susceptibility, evidence of a causal relationship is still lacking. The overexpression of the p53 gene has been shown to lead to excessive neuronal death and impaired neural function 14 . In patients with SCZ, p53 activation can enhance apoptosis 15,16 . Therefore, p53 hyper-activation may enhance tumor surveillance with higher neuronal apoptosis, which impairs psychiatric function. In addition, transcriptional regulation may help to explain the potentially causal relationship between tumor suppressor gene activity and SCZ 17 . It was also reported that anti-anxiety and anti-depressant drugs, such as midazolam and amitriptyline, have shown anti-cancer effects through p53-dependent cell cycle arrest and therefore, the induction of apoptosis 18 . However, a causal relationship between anxiety/depression and cancer, and the role of p53 in this relationship has not yet been elucidated 19 .
Therefore, we investigated the effects of p53 on anxiety and depression using p53 knockout mice treated with/without chronic unpredictable mild stress (CUMS) and possible mechanisms 20 .

p53 −/− mice show anxiety and depression under CUMS
We evaluated the effects of p53 knockout on anxiety-related and depression-related behaviors in p53 −/− mice. CUMS was performed as described 21 , and a behavioral test was shown in Figure 1A. In the anxiety test, p53 −/− mice performed signi cantly worse on the central zone time (%), entries, and distance (cm) in the open eld test (OFT) after CUMS ( Figure 1B). The performance of p53 −/− mice in the elevated zero maze (EZM), which also evaluates anxiety-like behaviors, was signi cantly lower than that of wild-type (WT) mice in time (%) and entries in the open arm after CUMS ( Figure 1C). Using the tail suspension test (TST) and forced swimming test (FST), depression-like behaviors were demonstrated through the increased immobility of p53 −/− mice after CUMS compared to WT mice ( Figure 1D and E). Together, these ndings indicate that a de ciency of p53 increases higher anxiety and depression. p53 de ciency under CUMS increases calcium and glutamate levels in PFC After the behavioral tests, we collected brain tissues from mice, and measured calcium levels in the prefrontal cortex (PFC). A signi cant increase of calcium in the p53 −/− PFC after CUMS compared to WT mice was shown by Alizarin Red S staining (Figure 2A). We next investigated the expression of calcium related signal proteins; calpain-1 and calpain-2. Calpains are mediators of calcium and neuroprotection in nerves, especially calpain-1, which is involved in neuroprotection, whereas calpain-2 facilitates neurodegeneration 22 . The mRNA and protein expression of calpain-1 was much signi cantly increased in WT mice after CUMS compared to p53 −/− mice after CUMS, but that of calpain-2 was signi cantly increased in p53 −/− mice after CUMS compared to WT mice after CUMS ( Figure 2B and 2C). Next, we measured the calcium-related neurotransmitter; glutamate by HPLC. Signi cantly increased levels of glutamate were found in the p53 −/− mice PFC after CUMS compared to WT mice PFC after CUMS ( Figure  2D). N-methyl D-aspartate receptor subtype 2A (NMDAR2A) and NMDAR2B are subtype of calciumpermeable ionotropic glutamate receptors in neurons. During pathological conditions, increased glutamate activate NMDAR and result in increased calcium in ux and neuronal death by preferential activation of NMDAR; NMDR2A is related to neuronal survival, whereas NMDAR2B is related to neuronal death signaling 23 . Recent studies suggest that NMDRAR2A de ciency cause schizophrenia-like phenotype 24,25 . Thus, we examined NMDAR2A and NMDAR2B expression by PCR and Western blot analyses in mice. NMDAR2B expression signi cantly increased in p53 −/− mice compared to WT mice PFC after CUMS, whereas the expression of NMDAR2A was signi cantly much increased in WT mice compared to p53 −/− mice PFC after CUMS ( Figure 2E and 2F). There were no signi cant differences in the calpain-1, calpain-2, NMDAR2A, and NMDAR2B expressions between p53 −/− and WT mice HP after CUMS (Supplementary Figure 1). These results suggest that signi cantly increased glutamate secretion and calcium in ux after CUMS in neurons was associated with calpain-2 and NMDAR2B pathway in p53 −/− mice PFC, and effects may be involved in neuronal cell death.
p53 de ciency under CUMS leads to neuronal cell death and activates related signals The in ux of calcium into cells is an important signal for cell death. We examined whether these differential calcium and glutamate levels could result in cell death. Signi cantly decreased neuronal cell number and increased cleaved caspase-3-stained areas were observed via cresyl violet staining ( Figure   3A) and IHC ( Figure 3B), respectively, in p53 −/− mice PFC after CUMS. However, there was no signi cant difference in the neuronal cell death between p53 −/− mice HP and WT mice HP (Supplementary Figure 2).
We evaluated the expression changes in the cell death relevant indicators through PCR and Western blot analyses. In line with the cell death pattern, the signi cantly increased expression of the caspase-3 ( Figure 4A) gene and cleaved caspase-3 protein was found ( Figure 4B) in p53 −/− mice PFC after CUMS.
Cell death-related protein (p-p38 and p-JNK) expression was also signi cantly increased in p53 −/− mice PFC after CUMS ( Figure 4C), whereas the expression of neuroprotective indicators (Wip1, brain-derived neurotrophic factor (BDNF), p-Akt, p-ERK, and p-CREB) was signi cantly decreased in p53 −/− mice PFC after CUMS compared to WT mice PFC after CUMS ( Figure 4D, 4E, and 4F). There were no signi cant differences in the expression of these signals in p53 −/− and WT mice HP (Supplementary Figure 3). These results indicate that p53 de ciency increase cell death through upregulating cell death signals and downregulating neuroprotective signals under CUMS.

P53 De ciency Under Cums Induces Neuroin ammation
To determine whether the cell death effect in p53 −/− mice is associated with the activation of astrocytes and microglia, the expression levels of GFAP (astrocyte activation marker) and Iba-1 (microglia activation marker) were detected through Western blot. A signi cant increase in GFAP and Iba-1 expression was observed ( Figure 5A). Next, we measured the release of in ammatory cytokines (IL-1β, IL-6, and TNF-α).
Signi cant increases in the gene mRNA expression of in ammatory cytokines in p53 −/− mice PFC after CUMS ( Figure 5B). Higher cytokines concentration in p53 −/− mice serum after CUMS were also observed compared to WT mice after CUMS ( Figure 5C). However, there were no signi cant differences between p53 −/− mice HP after CUMS and WT mice HP in the activation of microglia and astrocytes as well as cytokines releases (Supplementary Figure 4). These data indicate that a de ciency of p53 in mice under CUMS causes neuroin ammatory activity.
Effects of p53 inhibition on cell survival, glutamate/calcium levels, and cell death signals An in vivo study demonstrated that p53 de ciency increases cell death by increase of glutamate release and calcium in ux as well as cytokine release. To mimic the CUMS model in cells, PC12 cells were treated for 24 hours upon stimulation with corticosterone (CORT; 100 µM) with/without the inhibition of p53 by pi thrin-α (PTF-α; 100 µM). We observed that calcium levels were increased in CORT-treated PC12 cells via Alizarin Red S staining (red intensity increased) and further increased by the combination treatment of cortisone and PTF-α, but the cell number was decreased by CORT treatment and further decreased by the combination treatment ( Figure 6A). The expression of p53 was downregulated by CORT treatment and further decreased by PTF-α treatment. The expression of the calcium related calpain-1 -2 mRNA and protein were signi cantly increased by CORT treatment, but it was decreased by a combination treatment, whereas signi cant increase was observed in the calpain-2 by a combination treatment ( Figure 6B and 6C). Next, we measured the glutamate levels in the supernatant of PC12 cells by HPLC. Glutamate levels were signi cantly increased by CORT treatment and further increased by the combination treatment ( Figure 6D). Glutamate receptor subtype gene (NMDAR2A and 2B) and protein expression were detected.
The expression of NMDAR2A was increased by CORT treatment, but decreased by the combination treatment, whereas that of NMDAR2B was increased by CORT treatment and further increased by the combination treatment ( Figure 6E and 6F). We next investigated the cell death signal pathway. Caspase-3 gene mRNA expression increased signi cantly after CORT treatment, but CORT treatment with/without PTF-α was not signi cantly different ( Figure 7A), but the expression of cleaved caspase-3 was signi cantly increased by CORT treatment and further increased by PTF-α ( Figure 7B). The expression of cell death signal proteins (p-p38 and p-JNK) were also signi cantly increased by CORT treatment and further increased by the combination treatment with PTF-α ( Figure 7C). Conversely, CORT-induced the expression of neuroprotective signal proteins (Wip1, BDNF, p-Akt, p-ERK, and p-CREB) was increased, but was decreased by the combination treatment with CORT and PTF-α ( Figure 7D, 7E, and 7F). In ammatory cytokine (IL-1β, IL-6, and TNF-α) mRNA and release levels were signi cantly increased by CORT treatment and further increased by the combination treatment with PTF-α ( Figure 8A and 8B).

Discussion
Although several studies have demonstrated the inverse correlation between p53, a tumor suppressor gene, and ND susceptibility, a causal relationship between anxiety/depression and cancer has not yet been reported 11,14 . Several studies have demonstrated that p53 may serves to maintain important neuronal systems and synaptic functions 26,27 . It is also noteworthy that the absence of p53 activates the neuronal cell death pathway, which can lead to nerve damage in AD, PD, and SCZ 15,28,29 . Therefore, we speculated that the absence of p53 could worsen anxiety and depression. To solve out this speculation, we performed anxiety and depression behavioral tests in p53 −/− mice. Our data showed that the p53 −/− mice enhanced anxiety and depression behaviors after CUMS. Similar to our data, a recent study showed increased anxiety and depressive behaviors in WT p53-induced phosphatase 1 (wip1)-de cient mice 21 .
However, the mechanism how de ciency of p53 enhances anxiety and depression remains unclear.
Glutamate is ubiquitous within the CNS and has been shown to play important roles in many brain processes, including neurodevelopment (e.g., differentiation, migration, and survival) and learning (e.g., long-term potentiation and depression). However, higher levels of glutamate cause excitotoxicity associated with acute neurodegeneration (e.g., cerebral ischemia and traumatic brain injury), chronic neurodegeneration (e.g., HD and AD), and anxiety disorders 30 . The normal in ux of calcium is also important for maintenance of neuron functions. However, chronic calcium in ux is also associated with speci c neurotoxic events. A study showed that intracellular calcium concentrations are increased in patients with bipolar disorder and depression 31 . Moreover, calcium channel blockers prevent calcium in ux, and is used as an antidepressant. A study also showed that calcium channel blockers such as verapamil, diltiazem, and unarizine are effective at reducing anxiety-related behaviors 32 . Calcium channel blockers has been also proven effective at reducing depressive-like symptoms in both animal and human studies [32][33][34] . Our results showed an increase in the release of glutamate and calcium in p53 −/− mice brains after CUMS. This increase of glutamate and calcium causes was associated neuronal cell death. The type of glutamate receptor (NMDAR2A or 2B) determines neuronal cell death or neuroprotective activation 35 . While NMDAR2A is associated with neuroprotection, NMDAR2B is associated with apoptosis, and both receptors increase calcium in ux 36 . In addition, calpain, a calciumdependent protein, also functions differentially; calpain-1 is related to neuroprotection, while calpain-2 is related to cell death 37,38 . In the present study, we found that with glutamate release and calcium in ux, the expression of NMDAR2B and calpain-2 was increased, while the expression of NMDAR2A and calpain-1 was decreased in the p53 −/− mice brain after CUMS. We also found that cell death was increased in mice brains after CUMS and in CORT-treated PC12 cells, and PC12 cell death was further increased by the combination treatment of CORT and p53 inhibitor (PTF-α). These results indicate that the increase of glutamate and calcium levels through the differential expression of NMDAR2 and calpain may be associated with cell death in p53 −/− mice brains after CUMS and in CORT + PTF-α-treated PC12 cells. These results further indicate that glutamate-and calcium-dependent neuronal cell death could enhance anxiety and depression in p53 −/− mice.
Furthermore, calcium and glutamate have been associated with cell death signals (cleaved caspase-3, p-p38, and p-JNK) and neuroprotective factors (Wip1, BDNF, p-Akt, p-ERK, and p-CREB) 39,40 . Our animal study demonstrating the absence of p53 under stress increased cell death signals but decreased neuroprotective signals. Moreover, in cultured PC12 cells, CORT treatment increased cell death signals, which were further increased by PTF-α treatment. CORT-induced upregulated neuroprotective signals were inhibited by PTF-α. The elevation of glutamate and calcium could increase the release of cytokines 30 . In our study, we found that in p53 −/− mice brains after CUMS and in CORT-treated PC12 cells, IL-1β, IL-6, and TNF-α was signi cantly elevated, and PTF-α cotreatment further released these cytokines. It is noteworthy that neurons are vulnerable to in ammatory attacks 41 . Considering the signi cant involvement of in ammatory cytokines in patients with NDs, such as AD, PD, and HD 42 , elevation of these cytokine elevations could worsen anxiety and depression in p53 −/− mice after CUMS. Therefore, our data demonstrated that in p53 de cient mice and CORT-treated PC12 cells, the activation of cell death signals and enhanced cytokine release resulted in neuronal cell death.
Conclusively, we found that a de ciency of p53 could increase anxiety and depression by glutamate-, calcium-and cytokine-dependent neuronal cell death. p53 is a tumor suppressor gene, and it inhibits the development of many cancers, such as breast, gastric, and lung cancer 43 . Therefore, results suggest that p53 de ciency induces anxiety and depression, and these effects are inversely related to the p53dependent inhibition of cancer development.

Methods
All methods carried out in accordance with relevant guidelines and regulations.

Animals
The p53 -/-Tg mice (C57BL/6J-Trp53 em1hwl /Korl; KO created by TALEN-induced NHEJ) were thankfullygiven from the MISP (Osong, Korea). The non-transgenic and p53 -/-Tg mice used were C57BL/6 mice. The mice were housed and bred under speci c pathogen-free conditions at the Laboratory Animal Research Center of the Chungbuk National University, Korea. All experimental procedures in the present study were approved by the IACUC of Chungbuk National University (approval number: CBNUA-1277-19-01) and performed by ARRIVE guidelines. The mice were maintained in a room with a constant temperature of 22 ± 1 °C, relative humidity of 55 ± 10%, and under a 12/12-h light/dark cycle. Standard rodent chow (Samyang, Gapyeong, Korea) and puri ed tap water were available ad libitum.
CUMS procedure CUMS was performed as described in the references 21 . Brie y, as control mice were socially housed and undisturbed, mice for CUMS were singly housed and daily experienced a random stimulus of 10 for a speci ed duration. Stimuli were cold water swimming (13±1 °C, 5 min) (A), warm water swimming (37±2°C Tail suspension test TST was performed as described in the references 21 .Brie y, animals were suspendedabove the oor and recorded by a video camera for6 min. The duration of immobile behavior was manuallymeasured blinding to the treatment. The increase o mmobility indicated the depression-like behavior.

Forced Swim Test
FST was performed as described in the references 21 .Brie y, animals were placed in a cylindercontaining water and recorded by a video camera for6 min. The duration of climbing and immobile behaviorswas manually measured in the rst 2 min and last4 min respectively blinding to the treatment. The increaseof immobility and decrease of climbing both indicated thedepression-like behaviors.

Brain collection and preservation
After the behavioral tests, mice were perfused with phosphate-buffered saline (PBS, pH 7.4) under inhaled CO 2 anesthetization. The brains were immediately removed from the skulls and divided into the left brain and right brain. One stored at − 80 °C and the other xed in 4% paraformaldehyde for 48 h at 4 °C and transferred to 30% sucrose solutions.

Immunohistochemical staining
After being transferred to 30% sucrose solutions, brains were cut into 10μm sections by using a cryostat microtome (Leica CM 1850; Leica Microsystems, Seoul, Korea). After two washes in PBS (pH 7.4) for 10 min each, endogenous peroxidase activity was quenched by incubating the samples in 3% hydrogen peroxide in PBS for 20 min, and then two washes in PBS for 10 min each. The brain sections were blocked for 1 h in 1% bovine serum albumin (BSA) solution and incubated overnight at 4 °C with a rabbit polyclonal antibody against cleaved caspase-3 (1:100; Cell Signaling Technology,Danvers, MA, USA). After incubation with the primary antibodies, brain sections were washed three times in PBS for 10 min each. After washing, brain sections were incubated for 1-2 h at room temperature with the biotinylated goat anti-rabbit IgG-horseradish peroxidase (HRP) secondary antibodies (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Brain sections were washed thrice in PBS for 10 min each and visualized by a chromogen DAB (Vector Laboratories) reaction for up to 10 min. Finally, brain sections were dehydrated in ethanol, cleared in xylene, mounted with Permount (Fisher Scienti c, Hampton, NH), and evaluated on a light microscope (Microscope Axio Imager.A2, Carl Zeiss, Oberkochen, Germany).

Western blotting
In in vivo study, for comparing the expression of protein levels through Western blotting, we selected and used 4 of 10 mice brain from each group. Protein was extract by PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Inc., Seongnam, Korea). An equal amount of total protein (20 μg) was resolved on 8-15% sodium dodecyl sulfate polyacrylamide gel and then transferred to a nitrocellulose membrane . The blots were then incubated with the corresponding conjugated goat anti-rabbit or goat anti-mouse or donkey anti-goat IgG-horseradish peroxidase (HRP) (1:5000; Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA) secondary antibodies, 1 hours 30 min incubation at room temperature. Immunoreactive proteins were detected with an enhanced chemiluminescence Western blotting detection system. The relative density of the protein bands was measured by ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD).

Histochemical staining for calcium
Slides were then incubated in 2% Alizarin Red S (Sigma A-5533) pH 4.3 (adjusted with ammonium hydroxide) and blotted to remove excess dye. Slides were then dipped 20 times in acetone, followed by 20 times in acetone-Citrisolv. Slides were then cleared in Citrisolv a mountedslide evaluated on a light microscope (Microscope Axio Imager.A2, Carl Zeiss, Oberkochen, Germany).

Cresyl violet staining
Cresyl violet staining was performed as reported 44 . Frozen hippocampal tissues were cut into 10 μm sections by using cryostat microtome (Leica CM 1850; Leica Microsystems, Seoul, Korea). The pieces of tissues were xed in 4% paraformaldehyde for 24 h at 4 °C. In order to identify cortical layers and cytoarchitectural features of the isocortical region, the post-xed tissues were washed with PBS and then transferred to gelatin-coated slides and stained with 0.1% Cresyl violet (10 min). The sections were then washed with distilled water and dehydrated in 50%, 70%, 90%, and 100% ethanol for 2 min in each concentration. The tissues were airdried and immersed in a 1:1 mixture of absolute ethanol and xylene for 1 min. Following removal of the previous solution, the tissues were rinsed with xylene for 5-10 min and mounted with mounting medium (Cytoseal XYL, Thermo Scienti c, USA). The matching areas of tissues were photographed at 100xmagni cation.

RNA isolation and quantitative real-time RT-PCR
Tissue RNA was isolated from homogenized hippocampus using RiboEX (Gene All, Seoul, Korea), and total RNA (0.      The expression levels of GFAP and Iba-1in the mice PFC were detected by Western blotting using speci c antibodies.β-actin levels were measured to con rm equal protein loading. The values on the Western blot bands and graph represent the arbitrary density measured by ImageJ(A). The mRNA levels of IL-1β, IL-6, andTNF-α were detected by qRT-PCR in mice PFC (B). The protein levels of IL-1β, IL-6, andTNF-α were detected by ELISA in mice serum.Data are represented as means± SEM from three mice brains.*p<0.05Signi cant difference from WT + CUMS mice and #p<0.05 Signi cant difference from WT mice.   The mRNA levels of IL-1β, IL-6, andTNF-α were detected by qRT-PCR in PC12 cells (A