{'fraction_non_alphanumeric': 0.04237744089628762, 'fraction_numerical': 0.025477102256543344, 'mean_word_length': 4.752412160932096, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 1, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Human Herpesvirus 6B Greatly Increases Risk of Depression by Activating Hypothalamic-Pituitary -Adrenal Axis during Latent Phase of Infection

Summary Little is known about the effect of latent-phase herpesviruses on their host. Human herpesvirus 6B (HHV-6B) is one of the most ubiquitous herpesviruses, and olfactory astrocytes are one of the most important sites of its latency. Here, we identified SITH-1, an HHV-6B latent protein specifically expressed in astrocytes. Mice induced to produce SITH-1 in their olfactory astrocytes exhibited olfactory bulb apoptosis, a hyper-activated hypothalamic-pituitary-adrenal (HPA) axis and depressive symptoms. The binding of SITH-1 to the host protein calcium-modulating ligand (CAML) to form an activated complex promoted the influx of extracellular calcium. The serum antibody titers for depressive patients with respect to this activated complex were significantly higher than for normal controls (p = 1.78 × 10−15), when the antibody positive rates were 79.8% and 24.4%, respectively, and the odds ratio was 12.2. These results suggest that, in the latent phase, HHV-6B may be involved in the onset of depression.


HIGHLIGHTS
We identified SITH-1, a new protein specific to HHV-6B latent infection Mice expressing SITH-1 at HHV-6B latent infection site had depressive symptoms Depressive symptoms due to SITH-1 were associated with a hyper-activated HPA axis SITH-1-specific antibody detection significantly greater in depressive patients

INTRODUCTION
Herpesviruses have the property to maintain a state of latent infection in hosts throughout their lives and are an important component of the viral microbiome (virome), which occasionally increases the risk of various diseases (Virgin and Todd, 2011;Virgin et al., 2009).
Human herpesvirus 6B is a ubiquitous, neurotrophic virus that is widespread in many parts of the world, including the USA, Europe, and Japan. It causes roseola in the initial infection and afterward establishes latency. The virus is reactivated in response to immunosuppression in transplant patients and sometimes causes encephalitis (Ablashi et al., 2014).
Recent postmortem research revealed that HHV-6B and HHV-6A, a close relative of HHV-6B, had been reactivated from the latent state and had caused productive infection in the cerebellum in patients with mood disorders (major depressive disorder and bipolar disorder [BPD]) (Prusty et al., 2018). Although many aspects of the relationship between HHV-6A/B and these diseases were unknown, the authors stated the importance of conducting research on the mechanism by which latent HHV-6A/B are reactivated and infect the brain. Other past research reported detection of HHV-6B DNA in the orbital frontal cortex of patients with BPD (Conejero-Goldberg et al., 2003).
Proposed sites of HHV-6B latency are the tonsils and adenoids, and the virus has been shown to be actively shed in saliva. The shed virus enters the olfactory pathway and establishes latency in the astrocytes in the olfactory bulb/tract and nasal endothelium, other sites of HHV-6B latency (Ablashi et al., 2014;Donati et al., 2005;Harberts et al., 2011).
The olfactory bulb (OB) is not only a site of HHV-6B latency but also an immune organ that prevents virus in the saliva from invading the brain (Durrant et al., 2016). So, it is possible that the state of the OB is strongly associated with the reactivation of HHV-6B and invasion into the brain. OB dysfunction and a decrease in its volume have been reported in depressive patients (Negoias et al., 2010;Rottstaedt et al., 2018). We therefore considered that the effect of HHV-6B infection on the OB and other parts the olfactory system would differ between depressive patients and normal controls (NCs). As it has been reported that HHV-6B In order to identify astrocyte-specific HHV-6B latent protein, we used the latent protein of human cytomegalovirus (HCMV), a close relative of HHV-6B, as a reference. In our previous research, we identified two cytomegalovirus latent transcripts (CLTs) that code for latent proteins in HCMV. One of these CLTs (CLT ORF94) codes for latent protein open reading frame (ORF) 94, which consists of 94 amino acids, and the other (CLT ORF152) for ORF152, consisting of 152 amino acids (Kondo et al., 1996). In HHV-6B, we had already identified HHV-6 latent transcript (H6LT) type I and H6LT type II, homologs of CLT ORF94. They are expressed in macrophages and have the capacity to regulate reactivation of HHV-6B (Kondo et al., 1991(Kondo et al., , 2003, which suggests that they are related to HHV-6B latency in tonsils and adenoids. In the present research, we looked for a homolog of CLT ORF152. This led to identifying a new HHV-6B latent transcript that codes for a protein consisting of 159 amino acids, and we named it Small protein encoded by the Intermediate stage Transcript of HHV-6-1 (SITH-1) (Figures 1A and S1A-S1D). This protein had amino acid homology (20% identity, 74% similarity) with ORF152 (Kondo et al., 1996). We studied SITH-1 expression using macrophage (MF) cell lines (THP-1 and HL-60) and astrocyte cell lines (U373 and A172). Although no HHV-6B replication was observed (data not shown) in these cell lines, expression of latency-associated genes U94 (Rotola et al., 1998) and/or H6LT was detected (Figures S1E and S1F) so we considered them to be latency-like gene expressing cells. SITH-1 mRNA was mainly expressed in astrocyte cell lines (U373 and A172) ( Figure 1B). Also, SITH-1 protein was produced in HHV-6B-infected U373 cells without production of late proteins (glycoprotein [g]B and gH) ( Figure 1C). Based on these properties, SITH-1 was considered to be the latent protein produced during HHV-6B latency in olfactory astrocytes that we were looking for in this study.

Characterization of SITH-1 Expressing Mice
We examined the effects of SITH-1 expression in olfactory astrocytes in mice. We created the mouse model (SITH-1 mouse) through intranasal inoculation with an adenovirus vector (SITH-Ad) possessing the SITH-1 open reading frame (ORF) downstream of the glial fibrillary acidic protein (GFAP) promoter, which is specifically expressed in astrocytes. In the SITH-1 mouse, SITH-1 was produced in olfactory ensheathing cells (OECs), a type of olfactory astrocyte ( Figure S2A), and apoptosis occurred in the olfactory bulb ( Figures 1D-1F). The apoptosis in the olfactory bulb occurred mainly in astrocytes ( Figures S2B-S2D). As impairment of the olfactory bulb has been reported to be associated with depression (Morales-Medina et al., 2017;Rottstaedt et al., 2018), we subjected SITH-1 mice to the tail suspension test (TST) to see if they exhibited depressive symptoms. There was an increase in immobility time, indicating depression-like behavior, which was suppressed by an anti-depressant (SSRI) ( Figure 1G). This suggests that the SITH-1 mice were presenting depressive symptoms.
We next studied the SITH-1 mice to see if they manifested the pathological state associated with depression. Since it has been found that depression is induced by stress (Belmaker and Agam, 2008;Keller et al., 2017), we examined an association between SITH-1 mouse behavior and stress. When the mice were subjected to mild stress by tilting their cages, they exhibited decreased sucrose preference, a symptom of depression ( Figure 2A). Moreover, the expressions of corticotropin-releasing hormone (CRH) (Keller et al., 2017)  iScience Article (Ota et al., 2014), which is considered essential to depression induced by HPA axis activation, was also increased ( Figure 2D). Steroidogenic acute regulatory protein (StAR) (Otawa et al., 2007), the rate-limiting factor for corticosteroid production in the adrenal gland, was also up-regulated ( Figure 2E). Ratios of SITH-1 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown. (C) SITH-1 protein production in HHV-6B-infected U373 cells. Confocal imaging of HHV-6B infected and mock infected U373 cells. The upper six panels show staining with anti-SITH-1 antibodies and anti-gB antibodies and the lower six panels staining with anti-SITH-1 antibodies and anti-gH antibodies. Green, SITH-1; red, gB or gH; blue, DAPI.  rales-Medina et al., 2017). Considering these observations together, we believe that SITH-1 suppresses hippocampus neurogeneration via olfactory bulb impairment and, by doing so, activates the HPA axis, which results in the development of depressive symptoms. At this time, as there was no enhancement of inflammatory cytokine expression in the brain or olfactory bulb of the SITH-1 mice, the phenomena observed were considered not to be due to an immune response to SITH-1 ( Figures S3A-S3J). Also, to exclude the possibility that SITH-1 expression outside the olfactory system causes depression-like phenomena, we administered SITH-Ad at the same titer as for intranasal inoculation orally and intraperitoneally and examined behavior as well as gene expression. No depression-like behavior or increase in depressionrelated gene expression was observed ( Figures S4 and S5).

Functional Analysis of SITH-1
Next, we examined the mechanism by which SITH-1 induced apoptosis in astrocytes of the olfactory bulb. In twohybrid screening, human calcium-modulating cyclophilin ligand (CAML) was identified as a protein binding to SITH-1 (data not shown). The binding of SITH-1 and CAML was suggested by the mammalian two-hybrid system ( Figure 3A). CAML has been found to promote the influx of calcium into cells (Holloway and Bram, 1996). Also, it is believed to manifest this function through binding with other molecules in cells (Yamamoto and Sakisaka, . When SITH-1 was expressed in the human U373 astrocyte cell line, using confocal fluorescence microscopy, we observed the colocalization of SITH-1 and CAML as well as an increase in immunofluorescence intensity, suggesting accumulation of the SITH-CAML complex ( Figures 3B and S6). In addition, in the U373 cells in which SITH-1 was expressed, in the presence of extracellular calcium, there was an increase in intracellular calcium concentration ( Figures 3C and 3D). In view of this, the complex of SITH-1 and CAML was considered to have the property to induce influx of extracellular calcium into cells. Human CAML and mouse CAML (mCAML) have high homology (88% identity, 98% similarity), and the binding of SITH-1 and mCAML was suggested by the mammalian two-hybrid system ( Figure 3E). For further confirmation of the binding of SITH-1 and CAML determined using the mammalian two-hybrid system, we carried out a co-immunoprecipitation assay. Into HEK293T cells, which have hardly any endogenous CAML production, we transfected SITH-1 as well human or mouse CAML and carried out immunoprecipitation using anti-CAML antibodies. SITH-1 and CAML were co-immunoprecipitated, clearly showing that SITH-1 bound with both human and mouse CAML ( Figure 3F). In addition, SITH-1 brought about an increase in intracellular calcium concentration in mouse cells ( Figure 3G).
In order to confirm whether influx of extracellular calcium induced apoptosis in the olfactory bulb, we administered a calcium ionophore with a Ca 2+ transport enhancing action in the cell membrane into the nasal cavity of the mice. This resulted in enhancing apoptosis in the olfactory bulb ( Figure 3H). An increase in calcium concentration in OECs has caused ectopic neurovascular coupling in the olfactory bulb via release of ATP and glutamate (Thyssen et al., 2010), and by causing cerebrovascular stenosis (Mulligan and MacVicar, 2004) it could be associated with olfactory bulb cell impairment. In view of this, we consider that the mechanism by which SITH-1 induces apoptosis in the olfactory bulb is based on the promotion of extracellular calcium influx into OECs by the complex of SITH-1 and CAML.

Evidence for SITH-1 Expression in Depressive Patients
Next, we developed a method for investigating whether calcium influx due to SITH-1 occurs in vivo in depressive patients. Owing to the high invasiveness and difficulty of directly detecting SITH-1 expressed in the olfactory system, we decided to investigate the production of antibodies to the complex of SITH-1 and CAML. Based on a study using the SOSUI secondary structure prediction system (Hirokawa et al., 1998), the complex was assumed to be one with SITH-1 at the N terminus and CAML at the C terminus (N-SITH-CAML-C) ( Figures S7A-S7D). We measured intracellular calcium concentrations for cells expressing each of the structures shown in Figures S7A-S7D. As we had expected, N-SITH-CAML-C was strongest in promoting calcium influx, whereas a complex with CAML at the N terminus and SITH-1 at the C terminus (N-CAML-SITH-C) achieved hardly any calcium influx ( Figure 4A). Also, the strongest serum antibody reaction in the SITH-1 mice was with respect to N-SITH-CAML-C ( Figures 4B and 4C). Based on these observations, the antibodies produced in vivo in the SITH-1 mice to the SITH-1-CAML complex were anti-N-SITH-CAML-C antibodies. Thus, through measurement of these antibodies, it should be possible to study the formation of the SITH-CAML complex and calcium influx in vivo.
We next measured anti-N-SITH-CAML-C antibodies in depressive patients. Similar to the SITH-1 mouse, the serum antibody reaction in depressive patients was strongest with respect to N-SITH-CAML-C ( Figures  4D and 4E). Also compared with normal controls, the anti-N-SITH-CAML-C antibody titer was higher in depressive patients (p = 1.78 3 10 À15 ) ( Figure 4F). In analysis using receiver operating characteristic (ROC) curves, the area under the curve (AUC) was 0.8573, the antibody positive rates were 79.8% for depressive patients and 24.4% for healthy subjects, and the odds ratio was 12.2 (cutoff value 1.957) The use of Anti-N-SITH-CAML-C antibodies achieved diagnostic accuracy of 77.7% for depression ( Figure S7E). There was no difference between depressive patients and normal controls (NCs) regarding antibody titers for HHV-6 ( Figure S7F). Based on these findings, we considered that more SITH-CAML complex was being formed in depressive patients and that calcium influx was enhanced. Compared with NCs who had no depressive symptoms (Beck Depression Inventory [BDI] %3), the anti-N-SITH-CAML-C antibody titer was higher in NCs who had a few depressive symptoms (4 % BDI %10) ( Figure 4G). This finding may support the contribution of SITH-CAML to the risk of depression. There were no significant differences in sex ratio or age between depressive patients and NCs (Table S1). iScience Article

DISCUSSION
In the present research, we identified the HHV-6B latent protein SITH-1, and our findings in a mouse model in which SITH-1 was expressed in the olfactory astrocytes suggested that its binding with CAML enhanced calcium influx into cells and promoted OB apoptosis.
We also measured antibodies (anti-N-SITH-CAML-C antibodies) to the SITH-1-CAML complex in human serum. Differing from postmortem examination of brains, detection of antibodies is an indirect method Bars represent median values, and Kruskal-Wallis test with Steel's post hoc test (A, C, and E) and Mann-Whitney U-test (F and G). **, p < 0.01; ****, p < 0.0001.

OPEN ACCESS
iScience 23, 101187, June 26, 2020 7 iScience Article of evaluation, but it is minimally invasive and has the advantage of applicability as a test in patients who currently have depression. The detection rate of Anti-N-SITH-CAML-C antibodies in depressive patients was 79.8%, with a diagnostic accuracy of 77.7% for depression. In a previous study on antiphospholipid syndrome (APS), antibodies that react with the structure of active form b2-glycoprotein 1 were reported to be important for detection of APS (Pelkmans and de Laat, 2012). In the present study, the structure recognized by the detected antibodies was the SITH-1-CAML complex, which promotes calcium influx into cells, suggesting the possibility that they recognized a disease-related structure as in the case of APS.
Regarding an association with the OB, in imaging diagnosis, a decrease in OB volume was observed in depressive patients and diagnostic accuracy for depression based on this decrease was reported to be 68.1% (Rottstaedt et al., 2018). Thus, the diagnostic accuracy for depression using N-SITH-CAML-C antibodies in the present study and that using OB volume were close to each other, so it is conceivable that OB apoptosis due to SITH-1 expression similar to that in the mouse model occurs in depressive patients.
Using the mouse model, we found that promotion of OB apoptosis by SITH-1 activated the HPA axis. Since activation of the HPA axis has been observed to suppress host immune function by increasing corticosteroid production, its activation is thought to be advantageous for HHV-6B survival and reactivation. In addition, the OB is reportedly an immune organ that prevents viruses from invading the brain (Durrant et al., 2016) and induction of apoptosis in the OB may help HHV-6B in saliva to reach the brain. In this regard, a postmortem study reported proliferation of HHV-6B in cerebellums from depressive patients (Prusty et al., 2018).
We still know little about the cause of depression, and even in large-scale genetic analyses, no genes with an odds ratio of greater than 1.2 for an influence in depression have been discovered (Major Depressive Disorder Working Group of the Psychiatric GWAS Consortium et al., 2013;Levinson et al., 2014). In the results of the present study, at 12.2, the odds ratio for SITH-1-CAML complex antibody positivity having an influence in depression was extremely high. In view of this, multiple factors could be at work in the influence of HHV-6B and SITH-1 in depression.
HPA axis activation is known to raise depression risk (Belmaker and Agam, 2008;Keller et al., 2017). Also, the immunosuppression due to a hyper-activated HPA axis and decreased immune function caused by OB impairment would promote HHV-6B proliferation in the brain as suggested above. In addition, risks for depression have been reported for pathogens apart from HHV-6B, which include other herpesviruses and chlamydia (Wang et al., 2014). HPA axis activation and OB impairment may increase the risk of depression by facilitating the infiltration of these pathogens into the brain and their subsequent proliferation.
The mechanism by which SITH-CAML complex is produced in greater quantities in depressive patients than in normal controls will need to be studied further in the future. As a possibility, overwork is known to increase salivary HHV-6B (Aoki et al., 2016), and in this case, the increase in HHV-6B could bring about an increase in SITH-1-producing cells among OECs. A study finding that job strain was a risk factor for depression (Madsen et al., 2017) is considered to support this hypothesis.
We believe that the present study is the first to demonstrate an influence of a herpesvirus latent protein in a non-oncological disease. Similar to HHV-6B, other non-oncogenic herpesviruses also express (or likely express) latent proteins, so our methods would be applicable to research on the disease risk of other herpesviruses. We consider that our study suggests new directions for research on causes of depression as well as new perspectives for research on this group of viruses and the virome.

Limitations of the Study
When we examined SITH-1 expression in cells infected with HHV-6A and HHV-7, we were unable to detect SITH-1 mRNA with the structure indicated in this study. Additionally, in PCR-based studies (Okuno et al., 1995;Tanaka-Taya et al., 1996;Yoshikawa et al., 2000aYoshikawa et al., , 2000b and an immunological study (Wang et al., 1999), most Japanese were reported to be infected with HHV-6B. Therefore, we consider our present findings are thought to be limited to HHV-6B. However, if a latent protein with a similar function to SITH-1 can be identified for HHV-6A, it may be possible to extend the results of the present study to HHV-6A.

Materials Availability
All unique reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

METHODS
All methods can be found in the accompanying Transparent Methods supplemental file.

LEAD CONTACT AND MATERIALS AVAILABILITY
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Kazuhiro Kondo (kkondo@jikei.ac.jp). All unique reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

Viruses and cells
The macrophage cell lines THP-1 and HL-60 were cultured in Roswell Park Memorial Institute medium (RPMI 1640) containing 10% foetal bovine serum (FBS). The astrocytoma cell lines U373 and A172, as well as HEK293, HEK293T and 3T3 cells, were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS.
The HST strain of HHV-6B was used to infect the THP-1 and HL-60 cells at a multiplicity of infection (MOI) of 1, and the U373 and A172 cells at an MOI of 10, by centrifugation (37 °C, 2000 × g, 30 min). The recombinant adenovirus was produced using an adenovirus expression vector kit (Takara Bio) in accordance with the manufacturer's protocol. A pGfa2Lac plasmid incorporating the GFAP promoter was supplied by Dr. Kazuyoshi Ikuta. The GFAP promoter obtained from the pGfa2Lac plasmid and the SITH-1 gene amplified by PCR were cloned into an adenovirus cosmid vector using standard methods (Ad-GFAP-SITH1). HEK293 cells were transfected with the Ad-GFAP-SITH1 cosmid and a cosmid that did not contain the target gene (pAxcwit). The recombinant adenovirus was prepared in HEK293 cells and purified with an Adeno-X Virus Purification kit (Takara Bio). The purified virus titer was determined using an Adeno-X Rapid Titer kit (Takara Bio).

Subjects
The  (Table S1). However, the subjects in this 2 study did not have any symptoms suggestive of these conditions.
To investigate the relationship between stress and HHV-6B in saliva, the study enrolled 110 healthy volunteers (43.6 ± 0.8 [mean ± s.e.m.] years of age; 63 men, 47 women). They were recruited using an advertisement placed by Soiken Inc. Participants were excluded if they were currently taking any chronic medications or supplemental vitamins, or if they weighed less than 40 kg. They were also excluded if they had donated blood within a month of the study, or their blood haemoglobin level was less than 12.0 g/dL.
All human studies were approved by the Ethics Committees of the Jikei University School of Medicine, Tokyo Metropolitan Institute of Medical Science, Soiken Inc., and Soiken Clinic. Written informed consent was obtained from each subject.

Animals
Male 8

Identification of SITH-1 mRNA
We used a previously published HHV-6B latent infection system involving macrophage primary cultures (Kondo et al., 1991). Using the mRNA purified from these primary cultures, a reverse transcription reaction was performed with a random primer, IE4RB, as the reverse transcription primer of the sense transcript, and IE2FB as the reverse transcription primer of the antisense transcript. Next, the product of the reverse transcription (cDNA) was amplified by PCR with the primers IE4RB and IE2FB, and then by double-nested PCR with the inner primers IE4RA and IE2FA.  Figure S1.

Yeast two-hybrid screening
The Matchmaker Two-Hybrid System 2 (Clontech Laboratories) was used to screen for proteins that interact with SITH-1 protein following the manufacturer's protocol. SITH-1 was cloned into a pAS2-1 vector to produce a fusion protein of SITH-1 and a GAL4 DNA-binding domain (DNA-BD). SITH-1 was then amplified from SITH-1 cDNA by PCR. To investigate interactions with SITH-1, pAS2-1-SITH-1 was screened against the human foetal brain Matchmaker cDNA Library pACT2 (Clontech Laboratories) and Pretransformed Mouse 17-day Embryo Matchmaker cDNA Libraries (Clontech Laboratories) using a selective culture medium (without histidine, leucine, or tryptophan) in accordance with a standard protocol. Approximately 30 yeast clones were obtained and sequenced, and most were identified to be those including CAML or CAML fragments.

Mammalian two-hybrid assay
A Mammalian Matchmaker Two-Hybrid Assay Kit (Takara Bio) was used to confirm the binding of SITH-1 and CAML in mammalian cells. SITH-1 was cloned into a pM (GAL4) plasmid (pM-SITH-1). Human CAML and mouse CAML were cloned into pVP-16 plasmids (pVP-16-HsCAML, pVP16-MmCAML). Human CAML was amplified by PCR from the human foetal brain Matchmaker cDNA Library pACT2 (Clontech Laboratories).

Preparation of constitutively SITH-1-expressing U373 cells
A SITH-1-expressing recombinant retrovirus was prepared using the Retro-X Universal Packaging System (Takara Bio) in accordance with standard methods. SITH-1 was cloned into the retrovirus vector pQCXIP (pQC-4 SITH-1-IP). This vector can constitutively express the genes for SITH-1, IRES, and puromycin resistance under the control of the cytomegalovirus (CMV) early-stage promoter. The envelope vector p10A1 and either pQC-SITH-1-IP or pQCXIP (no insert) were co-transfected using GP2-293 packaging cells and the calcium phosphate method. The recombinant retrovirus was prepared in the GP2-293 cells, and the viral supernatant was recovered 48 h after transfection. U373-MG astrocytoma cells were infected with the recombinant retrovirus. After infection, the cells were treated with 1 µg/mL puromycin (Calbiochem) to select either constitutively SITH-1-expressing U373 cells (U373-SITH-1) or U373 cells infected with the retrovirus vector (no insert; U373-Vector).

Immunofluorescence staining of cultured cells
Rabbit anti-SITH-1 antibodies were made by inoculating rabbits with GST-tagged SITH-1 protein derived from expression using the pET -42a (+) vector system (Novagen). Stained HHV-6B infected U373 cells were prepared as described in the following. U373 cells were infected (MOI, 10) with HHV-6B using the centrifuge method. At After washes in PBS, Alexa Fluor 594 donkey anti-rabbit secondary antibodies (ThermoFisher) and Alexa Fluor 488 donkey anti-mouse secondary antibodies (ThermoFisher) were added, and the mixture reacted at 37 °C for 30 min. The fixed cells were then washed in PBS twice and mounted on slides. After cover glass application, samples were observed under the same optical conditions using a Carl Zeiss LSM880 confocal microscope equipped with Airyscan (Zeiss, Jena, Germany). The images were acquired by Zen black software (Zeiss).

Measurement of intracellular calcium concentration
U373 cells were first cultured on a glass slide. The fluorescent calcium reagent fura-2 acetoxymethyl ester (fura-2AM, Molecular Probes) was added to DMEM culture medium containing 10% FBS, and reacted at 37 °C for 30 min under dark conditions to allow uptake of the reagent. The cells were washed on the slide and then immersed in Hanks's Balanced Salt Solution with or without calcium. They were then stimulated with the calcium ATPase inhibitor thapsigargin (100 nM, Calbiochem). Images at excitation wavelengths of 340 nm and 380 nm were taken under a microscope with a CCD camera (IX71, DP70, Olympus) using Lumina Vision software (Mitani Corporation). The mean luminance of six cells was measured in ImageJ (NIH).
Regarding fusion proteins, HEK293T cells or 3T3 cells were cultured on a 96-well plate and transient overexpression of each protein for CAML, SITH-1, N-SITH-CAML-C and N-CAML-SITH-C was achieved for 24 h using Lipofectamine LTX (invitrogen) or Calphos Mammalian Transfection Kit (Clontech). Subsequently, cells were induced to take up Fluo 4-AM using Calcium Kit II-Fluo 4 (Dojindo) and then the fluorescence intensity in individual cells was measured using an ArrayScan XTI instrument (Thermo Fisher) (λex=480nm).

Immunoprecipitation and Immunoblot Analyses
HEK293T cells were transfected with SITH-1 and hCAML/mCAML expression plasmid DNA using the CalPhos

Mammalian Transfection Kit. After 24-h incubation, the cells were solubilized with M-PER Mammalian Protein
Extraction Reagent. For immunoprecipitation, mouse anti-CAMLG-Ab was immobilized on Dynabeads protein G. As a control, Dynabeads protein G without antibody was used. An equal volume of each cell lysate was incubated with Dynabeads at room temperature for 1 hour using a rotator. The beads were collected and washed with 0.2% Tween20/PBS. The collected beads and cell lysates were analysed by Western blot analysis with rabbit anti-SITH-1-Ab or rabbit anti-CAMLG-Ab.

Nasal inoculation of adenovirus vectors and tissue staining
For the nasal inoculation of adenovirus vectors, 8-week-old male C57BL/6 mice were anaesthetized with isoflurane. Recombinant adenoviruses were diluted in sterile water (not in isotonic buffer). A drop (20 µL) of SITH-Ad or Vector-Ad solution containing the virus at a titer of 1-2 × 10 9 plaque-forming units (pfu) /mL was placed at the entrance of the nasal cavity of the mouse. The solution entered the cavity through spontaneous respiration.
For the staining of the olfactory system, mice were sacrificed and transcardially perfused with saline, followed by 10% neutral buffered formalin (pH 7.4). Formalin-fixed tissues were then embedded in paraffin and sectioned. TUNEL staining was performed using the Apoptosis Detection Kit (Takara Bio) in situ. For immunofluorescence staining, paraffin-embedded sections were deparaffinized, and blocking was performed in Image-iT FX Signal Enhancer (Life Technologies) for 30 min. Primary antibodies were obtained from Abcam (active caspase-3 and GFAP) and Takara Bio (NSE). Secondary antibodies were obtained from Thermo (Alexa Fluor 488 Goat Anti-rabbit IgG (H+L), Alexa Fluor 594 Goat Anti-Mouse IgG (H+L)), Donkey anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 594). The antibodies were diluted with Can Get Signal Immunostain Solution A (TOYOBO). After the samples had been mounted on a slide with a cover glass, they were observed under a Keyence BZ-9000 fluorescence microscope.

Oral or intraperitoneal inoculation of adenovirus vectors
A sterile syringe was filled with one hundred µL of SITH-Ad or Vector-Ad solution containing the virus at a titer of 4 × 10 8 pfu/mL. Depending on the experiment, adenovirus vectors were administered to 8-week-old male C57BL/6 mice by oral gavage or intraperitoneal injection. The mice were bred for a week, examined for depression-like behaviour, and then the olfactory bulb, brain and adrenal glands were harvested.

Intranasal ionomycin administration
Ionomycin (FUJIFILM Wako) was dissolved in methanol to 1 mg/mL, and then diluted 10-fold with saline.
Eight-week-old mice were anesthetized, and a total 20 µL volume of 100 µg/mL ionomycin solution was dropped into the nasal cavity of each mouse. The control mice were administered saline containing 10% methanol. These mice were bred for 3 days, and then sacrificed.

Animal behaviour tests
Seven days after the inoculation of SITH-Ad or Vector-Ad, a tail suspension test ( To examine stress vulnerability, mice were intranasally inoculated with adenovirus vector and kept in a cage with a 20° tilt for 1 week (Harding et al., 2004). The sucrose preference test (Vincent et al., 2013) was used to examine depression-like behaviour. Mice had access to two bottles containing either 1% sucrose or water 3 days before recombinant adenovirus inoculation. After inoculation, each bottle was weighed daily to determine intake.

Real-time PCR
Total RNA was purified from cells using the BioRobot EZ1 and an EZ1 RNA Cell Mini Kit (Qiagen