TOB is an effector of the hippocampus-mediated acute stress response

Stress affects behavior and involves critical dynamic changes at multiple levels ranging from molecular pathways to neural circuits and behavior. Abnormalities at any of these levels lead to decreased stress resilience and pathological behavior. However, temporal modulation of molecular pathways underlying stress response remains poorly understood. Transducer of ErbB2.1, known as TOB, (TOB1) is involved in different physiological functions, including cellular stress and immediate response to stimulation. In this study, we investigated the role of TOB in the brain’s stress machinery at molecular, neural circuit, and behavioral levels. Interestingly, TOB protein levels increased after mice were exposed to acute stress. At the neural circuit level, functional magnetic resonance imaging (fMRI) suggested that intra-hippocampal and hippocampal-prefrontal connectivity were dysregulated in Tob knockout (Tob-KO) mice. Electrophysiological recordings in hippocampal slices showed increased postsynaptic AMPAR-mediated neurotransmission, accompanied by decreased GABA neurotransmission and subsequently altered Excitatory/Inhibitory balance after Tob deletion. At the behavioral level, Tob-KO mice show abnormal, hippocampus-dependent, contextual fear conditioning and extinction, and depression-like behaviors. On the other hand, increased anxiety observed in Tob-KO mice is hippocampus-independent. At the molecular level, we observed decreased stress-induced LCN2 expression and ERK phosphorylation, as well as increased MKP-1 expression. This study suggests that TOB serves as an important modulator in hippocampal stress signaling machinery. In summary, we show a molecular pathway and neural circuit mechanism by which TOB deletion contributes to expression of pathological stress-related behavior.


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On a daily basis, we encounter stressful events to which our bodies generate different responses 72 and store memories to cope with future occurrences. The brain utilizes several mechanisms to 73 cope with psychological stress, and defects in such mechanisms or exposure to excessive stress 74 can increase individual vulnerability to neuropsychiatric disorders like depression and post-75 traumatic stress disorder (PTSD) 1 . Strikingly, it is estimated that 50% of adults have 76 experienced a traumatic event during their lifetimes. Therefore, it is imperative that we 77 investigate mechanisms that underlie stress responses and identify potential therapeutic targets 78 coordinating stress resilience 2, 3 . 79 The stress coping response is orchestrated at various intercalated layers which include brain 80 connectivity, neuronal activity, molecular signaling, and resulting behavior 4 . Any change in 81 stress resilience mechanisms can induce psychiatric consequences, such as increased fear, 82 anxiety, and depression. Such behaviors are controlled by neuronal circuits governing 83 emotional and fight-flight responses, like the hippocampus, prefrontal cortex, amygdala, and 84 hypothalamus 5 . fMRI is currently the most advanced, non-invasive method to map dynamic 85 changes in brain circuits that regulate stress coping 6,7 . In response to stress, abnormal neuronal 86 circuit remodeling may occur, leading to altered brain connectivity. Several molecules have 87 been implicated in these remodeling events, like lipocalin-2 (LCN2) and corticotrophin-88 releasing factor (CRF) 8 . The Hypothalamic-Pituitary Adrenal (HPA) axis is a hormonal 89 signaling pathway that is moderately activated to elicit adaptation to induced stress at 90 molecular, cellular, physiological, and behavioral levels 9 . At the molecular level, acute stress 91 induces transcriptional and translation responses in order to cope with stress 10, 11 . This transient 92 change in molecular signaling is believed to have neuronal protective functions 12 . Our 93 knowledge of the hippocampal molecular stress machinery is limited; therefore, there are 94 continuing efforts to identify genes that function in stress coping responses 13 . Interestingly, 95 several molecules with known functions in cellular stress response have also been implicated 96 in psychological stress-coping mechanisms, e.g., EGR1 14,15 . 97 TOB has been proposed to regulate learning and memory, yet the mechanism is unknown 16, 17 . 98 Notably, Tob is one of the early response genes after either neuronal depolarization in 99 excitatory neurons or stress in humans 18,19 . In addition, TOB protein expression is elevated in 100 hippocampus and cerebellum after behavioral tests like fear conditioning and rotarod tests in 101 rats, respectively 16,17 . Moreover, decreased Tob gene expression has been correlated with 102 depression 20 . Taken together, this suggests that TOB participates in neuronal molecular 103 machinery and behavioral phenotypes. On the other hand, we previously showed that TOB 104 exhibits a unique transient elevation after exposure to UV stress, halting apoptosis, and then 105 eliciting an apoptotic signal after undergoing proteasome-dependent degradation 21 . In this 106 manner, TOB allows cells to recover through DNA repair mechanisms 22 . Furthermore, 107 overexpression of TOB in human bronchial epithelial cells leads to protection from ionizing 108 radiation-mediated cell death, increased ERK phosphorylation, and induced expression of 109 DNA repair proteins 23 . Stimulation using BMP-2, which induces oxidative stress, led to 110 increased TOB protein expression 24,25 . This suggests that TOB contributes to stress machinery, 111 mostly protective, at both the cellular and molecular levels. However, TOB's function in 112 psychological stress remains enigmatic. 113 Utilizing Tob-KO mice, we show that TOB has a functional role in stress coping behavior in 114 the brain by regulating hippocampal connectivity, neuronal excitability, and temporal 115 molecular changes induced by stress. Increased TOB protein expression in mouse brain after 116 exposure to acute stress, accompanied by the abnormal behavioral phenotype in 117 reveals TOB as key molecular effector in the brain's stress resilience. 118 119

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TOB protein increases in response to stress 121 TOB's function as an anti-proliferative protein is well known, but the potential role it plays in 122 regulating brain function is not well understood 16-18, 20, 26, 27 . With this objective, we analyzed 123 levels of TOB protein in mouse brain. We show that TOB is ubiquitously expressed across 124 various regions of mouse brain (Fig. 1A after mice were introduced to inescapable electric shock stress (Fig. 1D). ERK phosphorylation 136 levels also increased after acute stress, concurrently with TOB expression. Thus, TOB is 137 expressed in the mouse brain and its expression is increased following acute stress. 138 139 Deletion of Tob alters the brain's functional connectivity 140 Next, we sought to investigate the functional influence of Tob deletion on brain activity with 141 resting-state functional magnetic resonance imaging (rs-fMRI) in the awake state with 142 habituation to a small rodent MRI scanner. Awake resting-state fMRI for small animals allows 143 us to observe brain-wide synchronization of hemodynamic signals across multiple brain 144 regions. Prior to awake rs-fMRI sessions, Tob-KO and WT groups underwent habituation 145 training for 7 days in a mock MRI environment with MRI scanning sounds ( Fig. 2A receptor-mediated, paired-pulse facilitation was slightly increased in KO synapses ( Fig. 3F), 179 most pronouncedly at 10-ms inter-pulse intervals (p = 0.032). This suggested that TOB deletion 180 resulted in an increase in the number of mature postsynaptic AMPA receptors without changing 181 the AMPA receptor subunit composition. 182 We next investigated NMDA receptor-mediated synaptic transmission at CA3-CA1 synapses 183 in the hippocampus. We measured the input-output relationship and I-V curve of synaptic 184 NMDA receptor-mediated synaptic responses in the presence of the AMPA receptor antagonist, 185 NBQX. We found no apparent differences between genotypes ( Fig. S2 A We further examined inhibitory synaptic functions in hippocampal CA1 pyramidal neurons. 189 The amplitude (Fig. 3H), but not the frequency (Fig. 3I) of miniature inhibitory postsynaptic 190 currents (mIPSCs) were reduced in Tob-KO mice, compared to that of wild-type mice (Fig. 191 3G-I). These results indicate that TOB deletion affects both excitatory and inhibitory synaptic 192 transmission. 193 Next, we directly estimated the ratio of excitation to inhibition in hippocampal pyramidal 194 neurons. We first recorded AMPA receptor-mediated EPSCs at a holding potential of -60 mV, 195 equivalent to the Clequilibrium potential. Then we recorded  IPSCs at a holding potential of 0 mV, which is the reverse potential of AMPA and NMDA 197 receptors. We then calculated the ratio of EPSC amplitude to that of IPSC amplitude (E/I ratio) 198 and found that the E/I ratio was markedly increased in Tob-KO mice ( Fig. 3J-L). 199

Tob-KO mice show abnormal stress-related behavior 201
Contextual fear conditioning includes exposing mice to aversive acute stress caused by 202 inescapable electric shocks. Then brain regions respond by associating the context to such a 203 stimulus. Fearful mice freeze when exposed to the same context in which conditioning occurred. 204 On the other hand, contextual fear extinction is the subsidence of fear response due to repetitive 205 exposure to the same context without shock presentation 32 . After fear conditioning, response to an aversive context and decreased extinction, which was reversed by re-expression 215 of TOB. 216 The forced swim test, in which immobility is associated with increased despair, is widely used 217 to test depression-like behavior, but it is also an efficient test of the ability to cope with stress 33 . 218 Tob-KO mice showed increased immobility in the forced swim test (F3,28=13.50, p<0.0001; 219 WT vs KO p=0.0003). Re-expression of TOB in the hippocampus of Tob-KO mice reduced 220 immobility (KO(AAV_mTob) vs KO p=0.0008; WT(AAV_mTob) vs KO(AAV_mTob) 221 p>0.9999) (Fig. 4B). Similarly, we observed increased immobility by Tob-KO mice in the tail 222 suspension test, which was rescued by TOB overexpression in the hippocampus (Fig. S3E). 223 This shows that TOB in the hippocampus is important for coping with stress. 224 Since anxiety is usually observed in models showing abnormal stress coping mechanisms, we 225 next analyzed anxiety in our mouse model. difference between WT(AAV_mTob) and KO(AAV_mTob). Therefore, we believe that the 234 increased anxiety in Tob KO mice is not hippocampus-dependent. 235 In order to identify specific brain areas associated with Tob behavioral deficiencies, we 236 generated hippocampus-specific Tob-KO mice (hsTobKO) using the Cre-loxP system. First, 237 loxP sequences flanking exon2 were inserted in the Tob gene (Tob fl/fl ) (Fig. S4A to let their tails pass through. After restraint stress, mice were returned to their home cages for 430 indicated times, to be sacrificed for collection of hippocampi for protein extraction. 431 432 Inescapable electric shock 433 Mice were exposed inescapable electric shocks as described in the training procedure for "Fear 434 Conditioning and Extinction", and then returned to their home cages until sacrifice and 435 collection of hippocampi at the indicated times. 436 437

Functional magnetic resonance imaging (fMRI) 459
Detailed head-fixation bar mounting surgery and MRI imaging procedures are described in the 460 supplementary information. 461 462

Functional connectivity analysis 463
The pre-processed and denoised time series data were used for a seed-based FC analysis with 464 CONN17. Regions of interest (ROIs) including CA1, DG and mPFC were chosen. Seed-based 465 functional connectivity (FC) analysis was performed to compare FC between the Tob-KO 466 group and the control group. Seed-based FC analysis was composed of two steps. First, 467 Pearsons' correlation between a time series of an average seed ROI and each voxel in images 468 was calculated, and regional clusters were formed by thresholding statistical significance 469 (uncorrected p-value < 0.001) between two groups. In the second step, formed clusters were 470 further statistically corrected with a positive false discovery rate (pFDR; p < 0.05). 471 472

Electrophysiological recording 473
Electrophysiological recordings were performed as described by Etherton et al. 88 and are 474 detailed in the Supplementary materials and methods. 475 476

Quantitative real-time PCR 477
Total RNA was extracted from mouse hippocampi using Isogen II (Nippon Gene, Japan) 478 following the manufacturer's protocol. Reverse transcription was performed using PrimeScript 479 II 1st strand cDNA Synthesis Kit (Takara, Japan) following the manufacturer's protocol. Real-480 time PCR was performed using TB Green Premix Ex Taq II (Takara, Japan) and ViiA7 Real-481 Time PCR system (Applied Biosystems, USA). Relative mRNA expression was determined 482 by the ∆∆CT method and Gapdh mRNA levels were used for normalization. Primers used 483 were: 484 Gapdh FWD 5'-ctgcaccaccaactgcttag -3' REV 5'-gtcttctgggtggcagtgat -3'; Lcn2 Analysis was done using fastq files containing paired-end sequencing reads and analyzed using 502 nf-core/rnaseq pipeline v2.0 89 , which were mapped to the GRCm38 genome database using 503 STAR aligner (v2.6.1d) 90 . Mapped genes were then further analyzed using OmicsBox software 504 (v1.4.11) for counting using HTSeq (v0.9.0) 91 and differential gene expression analysis using 505 the package EdgeR (v3.11) 92 . Reads were normalized using the Trimmed Mean of M-values 506 (TMM) normalization method and a cut-off of at least 0.2 counts per million (CPM) in two 507 samples was selected. Differentially expressed genes (DEGs) were statistically tested using 508 EdgeR's exact test, and genes with FDR£0.05, p-value£0.05 and fold change (FC) ³2 or £-2 509 were used for further analysis. Pathway analysis was performed for genes 2-fold up-or down-510 regulated with p-value < 0.05 using Ingenuity Pathway Analysis (IPA) software (Qiagen, USA). 511 Raw and pre-processed transcriptomic data files described in the current study are publicly 512 available in NCBI GEO under accession number GSE186101.

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Behavior 515 Behavioral analyses were performed using male mice 8-12 weeks old. All experiments were 516 performed by experimenters blinded to genotype during testing. All software for analysis was 517 using an intraperitoneal injection of a mixture of Medetomidine (0.3 microgram/g), Midazolam 571 (5 µg/g) and butorphanol (5 µg/g). Additionally, a non-steroidal anti-inflammatory, Carpofen 572 (7.5 µg/g), was injected by the end of the surgery. Mice were fixed on a stereotaxic frame and 573 head hair was shaved. A 2% lidocaine solution was applied to the shaved skin and left for 2 574 min. Iodine was applied gently over the skin as an antiseptic. A midline incision was made, 575 and skin was retracted, and the skull was exposed. After drying the surface, the bregma was 576 detected. A micromanipulator was used to slowly move the injection needle to the target 577 injection site. A dental drill was used to drill a small hole, until the surface of the brain appeared. 578 A needle with viral solutions of around 300 nL was slowly advanced into the hole until it 579 touched the brain surface, and slowly lowered to the target coordinates. Injection was done 580 over 2 min and thereafter, the needle was left in place for 5 min before slowly retracting it. 581 Coordinates used for the CA1 region of the hippocampus were tested and optimized as 1.6 mm 582 posterior, 1.5 mm medio-lateral and 1.6 mm ventral to the bregma. 583 584 Immunohistochemistry 585 Immunohistochemical staining was performed as described by Matsuura et al., 2021 97 . 586 Antibodies used were Anti-Cre RpAb (Cell Signaling, USA) and Alexa Flour 488 Goat Anti-587 rabbit IgG (Invitrogen, USA). 588 589

Statistical analysis 590
All data are presented as means ± SEMs. T-tests, Mann-Whitney U test, one-way ANOVA, 591 and two-way ANOVA were used as described in figure legends. Multiple testing following 592 ANOVA was corrected using Bonferroni or Dunnet's post-hoc tests. GraphPad prism 9 was 593 used to perform all statistical analyses. 594