ATF6β Deficiency Elicits Anxiety-like Behavior and Hyperactivity Under Stress Conditions

Activating transcription factor 6 (ATF6) is an endoplasmic reticulum (ER) stress-regulated transcription factor that induces expression of major molecular chaperones in the ER. We recently reported that ATF6β, a subtype of ATF6, promoted survival of hippocampal neurons exposed to ER stress and excitotoxicity, at least in part by inducing expression of calreticulin, an ER molecular chaperone with high Ca2+-binding capacity. In the present study, we demonstrate that ATF6β deficiency in mice also decreases calreticulin expression and increases expression of glucose-regulated protein 78, another ER molecular chaperone, in emotional brain regions such as the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala. Comprehensive behavioral analyses revealed that Atf6b−/− mice exhibit anxiety-like behavior in the light/dark transition test and hyperactivity in the forced swim test. Consistent with these results, PFC and hypothalamic corticotropin-releasing hormone (CRH) expression was increased in Atf6b−/− mice, as was circulating corticosterone. Moreover, CRH receptor 1 antagonism alleviated anxiety-like behavior in Atf6b−/− mice. These findings suggest that ATF6β deficiency produces anxiety-like behavior and hyperactivity via a CRH receptor 1-dependent mechanism. ATF6β could play a role in psychiatric conditions in the emotional centers of the brain.


Introduction
Disruptions of intracellular environments, such as Ca 2+ dysregulation, energy shortage, and aberrantly increased protein synthesis, cause accumulation of unfolded proteins in the endoplasmic reticulum (ER), which is known as ER stress [1]. Cells respond to this type of stress by activating the unfolded protein response (UPR), which is regulated by at least three different branches, including protein kinase R-like ER kinase, inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6) [2]. In the central nervous system (CNS), ER stress is associated with pathologies such as brain ischemia, neurodegeneration, excitotoxicity, and neuroinflammation [3][4][5]. Further, recent reports have demonstrated that ER stress and the UPR play a role in physiological brain functioning. Wolfram syndrome 1 (WFS1) and Sigma-1 receptor (Sigma-1R) are both ERresident proteins that play critical roles in Ca 2+ homeostasis [6,7]. Deletion of Wfs1 or Sigmar1/Oprs1 genes in mice induces ER stress, impairs behavioral adaptation in stressful conditions [8,9], and induces depressive-like phenotypes [10]. Moreover, X-box binding protein 1, a major regulator of the IRE1 branch of the UPR, plays a regulatory role in memory formation [11].
ATF6 is an ER-resident type 2 transmembrane protein that transduces UPR signaling [12,13]. In response to ER stress, ATF6 migrates from the ER to the Golgi, where proteases cleave ATF6 to release the cytosolic, N-terminal ATF6 transcriptional activator fragment [14]. In mammals, there are two ATF6 subtypes, ATF6α and ATF6β, with ATF6α playing the predominant role in the UPR.
We previously reported that ATF6α contributes to neuronal survival and astroglial activation in neuropathological conditions such as brain ischemia, excitotoxity, and a Parkinson's disease model [15][16][17], although ATF6α enhances microglia-mediated neuroinflammation in experimental autoimmune encephalomyelitis [18]. Recently, we reported that calreticulin (CRT), an ER molecular chaperone with high Ca 2+ -binding capacity, is a unique target of ATF6β in the CNS, and that the ATF6β-CRT axis contributes to survival of the hippocampal neurons under ER stress and excitotoxicity by improving brain intracellular Ca 2+ homeostasis [19]. Although ATF6β is widely expressed in the brain and contributes to neuronal survival, its physiological role remains incompletely understood. Furthermore, during the regular maintenance of mice, including cage changing, we observed that ATF6β-deficient (Atf6b -/-) mice were more active than wild-type (WT) mice.
Therefore, in the present study, we tested the hypothesis that ATF6β regulates emotional behaviors in mice, revealing that ATF6β deficiency decreased CRT expression and increased expression of glucose-regulated protein 78 (GRP78), a major ER molecular chaperone, in emotional brain regions of mice. Comparative analyses between WT and Atf6b -/mice revealed that ATF6β deficiency elicited anxiety-like behavior, which was due to increased brain levels of corticotropin-releasing hormone (CRH) and increased circulating corticosterone. Moreover, CRH receptor 1 (CRHR1) antagonism alleviated anxiety-like behavior in Atf6b -/mice. These results suggest ATF6β deficiency elicits anxiety-like behavior and hyperactivity via a CRHR1dependent mechanism.

Animals
All animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals and were approved by the institutional review committee of Kanazawa Medical University (Approval No. 2018-21) and by the Animal Care and Use Committee of Kanazawa University (Approval No. AP-184,013). Experiments were conducted in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, and in compliance with ARRIVE guidelines. Atf6b +/mice were generated as previously described [20], and backcrossed to the C57BL/6 N strain for eight or more generations at the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University. Atf6b +/mice were intercrossed to obtain Atf6b +/+ , Atf6b +/-, and Atf6b -/mice. Atf6b +/+ mice were used as WT controls. The mice were each housed as littermate groups in a standard cage on a 12-h light/dark cycle (lights on at 7:00 AM) with food and water available ad libitum in the Institute of Experimental Animal Sciences, Kanazawa Medical University. A total of 65 male WT and 56 male Atf6b -/mice (age 11-21 weeks) were used for experiments.

In Situ Hybridization
In situ hybridization was performed as described previously [21]. Briefly, a − 600 bp mouse ATF6β cDNA fragment was polymerase chain reaction (PCR)-amplified using primer sets ( Table 1) and inserted into a pGEM-T vector (Promega, Madison, WI, USA). The plasmid was linearized using EcoRV and ApaI to synthesize the antisense and sense probes, respectively. Brains were isolated from mice after perfusion with phosphate-buffered saline (PBS) and immediately placed at -80℃. Serial 14-µm sagittal sections were generated using a cryostat and hybridized with a digoxigenin-labeled ATF6β RNA probe. Imaging was performed using a light and fluorescence microscope (BZ-X700, Keyence, Osaka, Japan).

Quantitative Real-time PCR (RT-qPCR)
Brain slices (1 mm-thick) were prepared using a coronal mouse brain slicer (Visikol, Hampton, NJ, USA) from the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala, identified using the mouse brain atlas. Total RNA was extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. RNA was then reverse transcribed to synthesize first-strand cDNA using a ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). RT-qPCR was then performed using oligonucleotide primer sets ( Table 2). Individual cDNA samples were preheated at 98 °C for 2 min and subjected to 40 amplification cycles (denaturation at 98 °C for 10 s, annealing at 60 °C for 10 s, extension at 68 °C for 30 s) using Thunderbird™ SYBR qPCR Mix (Toyobo) or KOD SYBR ® qPCR Mix (Toyobo), and a QuantStudio™ 12 K Flex Real-Time PCR System (Thermo Fisher Scientific, Tokyo, Japan). Cycle threshold values were calculated using the ΔΔ cycle threshold method to obtain fold differences. Relative expression of each gene was normalized against Gapdh mRNA expression level.

Behavioral Tests
All behavioral tests were performed between 8 and 11 a.m. with male mice (11-14-week-old). After each test, all testing apparatuses were cleaned with hypochlorous water to remove olfactory cues. All behavioral tests were performed at least 1 day apart.

General Health and Neurological Screen
A general health check and neurological screen was conducted as described previously [22]. Several physical features, including the presence of whiskers and bald patches, were recorded. The righting reflex and whisker and ear touch reflexes were also evaluated. In addition, body weight was measured, and neuromuscular strength was assessed using wire hang tests. In the wire hang test, mice were individually inverted, placed on a wire mesh with 5-mm square holes, and the latency to fall from the wire was recorded with a 60 s cutoff time.

Light/Dark Transition Test
A light/dark transition test was performed as previously described in two chambers [23], a light arena (25 × 25 × 24 cm, 485 lx) and a dark arena (16 × 25 × 24 cm, 6 lx). Individual mice were placed in the dark chamber and allowed to move freely between the two chambers for 10 min. The total number of transitions between chambers, the time spent in the light chamber (%), and the latency to first entrance into the light chamber (sec) were recorded automatically by a floor-pressure sensor and analyzed using software (PPCWIN v2.0, Panlab Harvard Apparatus, Barcelona, Spain).

Open Field Test
An open field test was used to evaluate locomotor activity and emotional response [24]. The open field test was measured with a circle field apparatus (45 cm height × 79 cm diameter), a central field (50%), and an outer field (periphery). Individual mice were placed in the periphery of the field at the start of the test and allowed to freely move about the apparatus for 10 min. The distance traveled and time spent in the center field (%) were recorded automatically Table 2 List of RT-qPCR primers used in this study. Table 1 List of in situ hybridization primers used in this study.

Rotarod Test
The rotarod test was performed to evaluate motor coordination and balance ability using an accelerating rotarod (Panlab Harvard Apparatus). Mice were placed on a rotating rod (3 cm diameter) that accelerated from 4 to 40 rpm over a 5-min period, and the time individual mice were able to maintain balance on the rotating rod was recording. Timing stopped when the mouse fell off the rotating rod (sec).

Porsolt Forced Swim Test
The Porsolt forced swim test [28] was performed as previously described [29]. The apparatus, consisting of an acrylic cylinder (50 cm height × 24 cm diameter), was filled with water at 23 °C up to a height of 20 cm. Individual mice were placed into the cylinders, and total distance and immobility time were recorded for 10 min (days 1 and 2) by video tracking software (SMART v3.0, Panlab Harvard Apparatus). The immobility time was set to 2.5 cm/sec or less for mouse movement.

Elevated Plus Maze Test
An elevated plus maze test was conducted as previously described [25]. The elevated plus maze consisted of two open arms (30 × 6 cm) and two enclosed arms with walls (30 × 6 × 13.5 cm). The arms and a central platform

Social Interaction Test
A social interaction test was conducted as described previously [26]. The social interaction test was performed in a gray plastic box (40 × 40 × 40 cm), and a wire cage (9 cm height × 9 cm diameter) with 6-mm square holes as the interaction apparatus was placed in one corner of the arena for the test. The interaction zone was defined as a 20-cm quadrant area surrounding the interaction apparatus, and the area diagonal to the interaction zone was defined as the evacuation zone. The area between the interaction and evacuation zones was defined as the interference zone. The day before testing, test mice were individually placed in the box without a stranger mouse (C57BL/6 N male) in the wire cage and allowed to freely explore the box for 10 min. In the social interaction test, a stranger mouse that had no prior contact with the test mouse was placed in the wire cage. The test mice were individually placed in the interference zone of the arena and allowed to explore freely for 10 min. The number of the contact zone entries, time spent in the contact zone (%), and the mean duration of time in the contact zone (sec) were measured automatically by video tracking software (SMART v3.0, Panlab Harvard Apparatus).

Hot Plate Test
The hot plate test was performed to evaluate sensitivity to a painful stimulus. In the hot plate test, mice were placed on a 55.0 °C hot plate (UGO Basile, Comerio, Italy) in an acrylic cylinder (height, 24.5 cm; diameter, 19 cm), and latency to the first paw response was recorded with a 15 s cutoff time [27]. The paw response was defined as either a foot shake or a paw lick. equal variances, and Welch's t-test was used when there was normality but no equal variances. One-way analysis of variance (ANOVA) followed by the Tukey-Kramer test were used for multigroup testing. Rotarod scores were compared using repeated-measures two-way ANOVA followed by the post hoc Bonferroni test. EZR on R commander version 1.55 was used for statistical analyses. P-values < 0.05 were considered statistically significant.

Expression of ATF6β and Molecular Chaperones in the Brain
First, we assessed ATF6β expression in the adult brains. Consistent with our recent report [19], in situ hybridization revealed that Atf6b mRNA was widely expressed in the brain of WT mice, with relatively higher levels in the hippocampus and cerebellum, whereas its expression was almost abolished in Atf6b -/mice (Fig. 1A). RT-qPCR revealed that Atf6b mRNA was almost completely lost in Atf6b -/mice in regions associated with emotional processing, including the PFC, hypothalamus, hippocampus, and amygdala (Fig. 1B). Expression of Calr (CRT), a molecular chaperone in the ER with a high Ca 2+ binding capacity, was significantly decreased in Atf6b -/mice (Fig. 1C), while expression of Hspa5 (GRP78), a major molecular chaperone in the ER, was increased in Atf6b -/mice (Fig. 1D) in all brain regions, as described in our recent report [19]. Consistently, paraventricular hypothalamic nucleus (PVN) CRT expression was lower in Atf6b -/mice than in WT mice (Fig. 1E, F).

Behavioral Characteristics of ATF6β-deficient mice
To investigate the physiological role of ATF6β in the brain, we performed comprehensive behavioral analyses using WT (Atf6b +/+ ) mice and Atf6b -/littermates. There were no obvious differences in general health and neuronal screening, including coat and whisker condition, whisker and ear touch reflexes, and the righting reflex, between WT and Atf6b -/mice. However, the body weight of Atf6b -/mice was lower than that of WT mice ( Fig. 2A). Muscle strength, which was measured by wire hanging, did not significantly differ between WT and Atf6b -/mice ( Fig. 2A).
We next examined anxiety-related behavior in Atf6b -/mice using the light/dark transition test, the open field test, and the elevated plus maze test. In the light/dark transition test, Atf6b -/mice exhibited significantly longer latencies prior to light box entry, decreased transition numbers, and decreased time spent in the light box (Fig. 2B). In the open field test, there were no differences in total distance traveled anti-CRH (1:2,000, Peninsula Laboratories, T-5007.0050, RRID: AB_518256). Anti-rabbit Alexa Fluor 488 (1:1,000, Molecular Probes, A-21,206, RRID: AB_141708), and antiguinea pig Alexa Fluor 488 (1:1,000, Molecular Probes, A-11,073, RRID: AB_142018) secondary antibodies were used to visualize immunolabeling. Imaging was performed using a laser scanning confocal microscope (Eclipse TE200U, Nikon, Tokyo, Japan) at 20X magnification with Nikon EZ-C1 software.
For quantification of immunohistochemical results, eight serial brain sections were selected per mouse. The intensities of CRT and CRH signals were calculated using ImageJ software (National Institutes of Health). Analyses were conducted blindly.

ELISA
To measure plasma corticosterone concentrations, we used a Corticosterone Enzyme Immunoassay Kit (K014: Arbor Assays, Ann Arbor, MI, USA) according to the manufacturer's instructions. Blood was collected from the tail veins of WT and Atf6b -/mice (14-16 weeks of age) under basal conditions, immediately combined with EDTA (1.4 mg/ ml), and centrifuged at 1,000 g for 15 min. The supernatant was stored at -80 °C until use. The dissociation reagent was allowed to warm completely to room temperature prior to use. Plasma was added to an equal amount of the dissociation reagent, vortexed gently, and incubated at room temperature for 5 min. The mixture was then diluted with assay buffer (1:100). Standard solutions with known corticosterone concentrations and plasma samples were pipetted into wells on a plate. DetectX® corticosterone conjugate was added to each well, and then DetectX® corticosterone antibody was added to each well except for the non-specific binding wells. The plate was covered with a plate sealer and placed on a shaker at room temperature for 1 h. After washing each well four times with wash buffer, the TMB substrate was added, and the plate was incubated at room temperature for 30 min without shaking. Subsequently, stop solution was added to each well, and absorbance values were read at 450 nm in a microplate reader (Multiskan GO, Thermo Fisher Scientific). The corticosterone concentration for each sample was calculated using the microplate reader software (Thermo Fisher Scientific).

Statistical Analyses
All data are presented as means ± SEM and individual values. In the test between two groups, the Kolmogorov-Smirnov test was performed for normality tests, and the F-test was performed for comparisons of two variances. Student's t-test was used when there was normality and We further examined the social behavior of Atf6b -/mice. In the social interaction test, the behavior of WT and Atf6b -/mice did not differ (Fig. 3A). Moreover, we assessed other physiological conditions in Atf6b -/mice using two behavioral analyses. The hot plate test was performed to evaluate sensitivity to a painful stimulus. There was no difference between WT and Atf6b -/mice in the latency to first paw response on the hot plate (Fig. 3B). The rotarod test was and time spent in the center zone between WT and Atf6b -/mice (Fig. 2C). In the elevated plus maze test, there were no differences in the number of transitions in each zone, the number of entries to the open arms, and the time spent in the open arms between WT and Atf6b -/mice (Fig. 2D). These results suggest that Atf6b -/mice exhibited anxiety-related behavior, especially anxiety about bright places.

CRH and Corticosterone Levels in ATF6β-deficient mice
To investigate in greater detail the role of ATF6β in the HPA axis, we compared Crh mRNA levels between WT and Atf6b -/brains. RT-qPCR revealed that PFC and hypothalamic Crh mRNA levels were significantly higher in Atf6b -/mice than in WT mice (Fig. 4A). Consistently, hypothalamic CRH expression in the PVN was higher in Atf6b -/mice than in WT mice (Fig. 4B, C). Further, the level of plasma corticosterone, a downstream molecule of the HPA axis [32,33], was significantly higher in Atf6b -/mice under basal conditions (Fig. 4D). Together, these data suggest that the HPA axis was more activated in Atf6b -/mice than in WT mice, similar to what was observed in mice exposed to chronic stress, which could result in anxiety phenotypes. After finishing all behavioral tests, however, Crh mRNA levels were not significantly different between WT and Atf6b -/brains, only slight tendency to be higher in Atf6b -/brains (Supplementary Fig. 1).

CRH Dependency of Anxiety-like Behavior in ATF6βdeficient Mice
To determine if anxiety-like behavior in Atf6b -/mice was dependent on CRH, the CRHR1 antagonist Antalarmin (10 mg/kg i.p.) or 4% DMSO vehicle control were injected into WT and Atf6b -/mice 40-50 min prior to behavioral testing [30,31] (Fig. 5A). The body weight of Atf6b -/mice was lower than that of WT mice with or without Antalarmin administration (Fig. 5B). Muscle strength did not differ performed to evaluate motor coordination and balance ability, but the latency to fall was not significantly different between WT and Atf6b -/mice (Fig. 3C). Contrastingly, in the forced swim test, the total distance traveled was significantly longer in Atf6b -/mice than WT mice, and the immobility time on day 2 was significantly shorter in Atf6b -/mice than WT mice (Fig. 3D). We think this is unlikely to be due to impaired memory function, because recently we reported that hippocampal memory function, as evaluated using the Morris water maze test, was not affected in Atf6b -/mice [19]. Collectively, these results indicate that ATF6β-deficient mice exhibited anxiety-like behaviors in approaching bright places, and hyperactivity under stress situations.

Stress Status and ATF6β Expression After Behavioral Analysis
To understand the relationship between stress status and ATF6β expression, we analyzed the transcription levels of Atf6b and Crh, a key factor in stress-induced responses in the HPA axis, using samples from different regions of the brain of WT mice, before and after finishing all behavioral tests. RT-qPCR revealed that the expression of Crh was significantly increased in the PFC and hypothalamus after behavioral tests (Fig. 3E). By contrast, that of Atf6b was significantly decreased in the hypothalamus under the same condition (Fig. 3F). Furthermore, the expression of Hspa5 was increased in the hypothalamus, hippocampus, and amygdala after behavioral tests (Fig. 3G), These results suggest a relationship between stress status, ATF6β expression, and UPR activation in the hypothalamus after behavioral analysis. to WT, and CRHR1 antagonism normalized anxiety-like behavior in Atf6b −/− mice, indicating that ATF6β deficiency elicits anxiety-like behavior, especially in the bright area, via CRHR1. To our knowledge, this is the first report demonstrating a role for the ATF6 branch of the UPR in behavior.

Discussion
The major findings of this study are that ATF6β ablation in mice caused anxiety-like behavior in the light/dark transition test, and hyperactivity in the forced swim test. PFC and hypothalamic CRH were increased in Atf6b −/− mice relative promotes CRHR1 activation remains incompletely understood, prior findings, together with the present findings, point to a few potential mechanisms. First, enhanced levels of neuronal activities in the emotional brains, including the hypothalamus, could be caused by Ca 2+ dysregulation alone, or in combination with ER stress. This could increase stimulation of HPA axis-associated neurons. We previously reported upregulation of neuronal activity-associated genes such as Igf2 [35], Igfbp2 [36], and Egr1 [37] in the hippocampus of Atf6b −/− mice [19]. Second, ATF6β may regulate the synthesis of CRH in the hypothalamus; this may explain why the expression of Crh significantly increased, while that of Atf6b significantly decreased in the hypothalamus after behavioral analysis (Fig. 3E, F). Similarly, the activation status of the UPR in the hypothalamus may also have an impact on the synthesis of CRH (Fig. 3G). Finally, glial cells, especially microglia, could also affect CRHR1 activation. For example, FK506 binding protein 51, a cochaperone of the glucocorticoid receptor (GR) in both neurons and microglia, increases GR resistance, leading to prolonged activation of CHRH1 [38,39]. Further studies using brain region and cell type-specific conditional Atf6b knockout will be performed to identify the physiological genes, both of which encode ER-resident proteins important for Ca 2+ homeostasis, induces ER stress and behavioral abnormalities in mice [8][9][10]. These observations suggest a role for ER stress and/or Ca 2+ dysregulation in behavioral abnormalities. However, Wfs1 −/− , Oprs1 −/− , and Atf6b −/− mice exhibit some phenotypic differences. Wfs1 −/− and Oprs1 −/− mice exhibit depression-like behavior, as indicated by longer immobility times in the forced swim test [8,10]. Contrastingly, in the present study, Atf6b −/− mice were hyperactive, as indicated by longer traveling distances and shorter immobility times in the forced swim test (Fig. 3D).
In addition to the effect of ER stress, dysregulation of Ca 2+ homeostasis alone could cause behavioral abnormalities in Atf6b −/− mice. Deletion of CD38, which mobilizes Ca 2+ from the ER by converting nicotinamide adenine dinucleotide to cyclic adenosine diphosphoribose, impairs social behavior in mice via dysregulation of hypothalamic oxytocin neurons [34]. However, in the present study, social interaction was not impaired in Atf6b −/− mice, suggesting that dysregulation of hypothalamic oxytocin neurons did not contribute to the observed phenotypes.
The present study identified an important role for CRHR1 activation in the anxiety-like behavior of Atf6b −/− mice. Although the mechanism by which ATF6β deficiency
The behavioral phenotypes observed in Atf6b −/− mice were relatively mild, and restricted to specific conditions such as light/dark transition and forced swim. It is quite important to investigate the responses to different types of stresses, either acute or chronic stresses. Furthermore, all experiments in this study were performed using male mice. It was recently reported that local production of CRH in PFC modulates male-specific novelty exploration [40]. In our preliminary experiments, ATF6β-deficient female mice are likely to show different phenotypes from those of male mice in behavioral tests, which will require further analysis.
In conclusion, we demonstrated that ATF6β-deficient mice exhibited CRHR1-dependent anxiety-like behaviors in the light/dark transition test and hyperactivity in the forced swim test. The findings suggest that these mice could be used to study the role of ER stress and the UPR in the neuropsychiatric and neuroendocrine research fields. Takashi

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflict of Interest
The authors declare no competing financial interests.