Mgat5 modulates the effect of early life stress on adult behavior and physical health in mice

Accepted Manuscript Title: Mgat5 modulates the effect of early life stress on adult behavior and physical health in mice Author: Laura Feldcamp Jean-Sebastien Doucet Judy Pawling Marc P. Fadel Paul J. Fletcher Robert Maunder James W. Dennis Albert H.C. Wong PII: DOI: Reference:

S0166-4328(16)30388-6 http://dx.doi.org/doi:10.1016/j.bbr.2016.06.033 BBR 10276

To appear in:

Behavioural Brain Research

Received date: Revised date: Accepted date:

13-2-2016 5-6-2016 15-6-2016

Please cite this article as: Feldcamp Laura, Doucet Jean-Sebastien, Pawling Judy, Fadel Marc P, Fletcher Paul J, Maunder Robert, Dennis James W, Wong Albert H.C.Mgat5 modulates the effect of early life stress on adult behavior and physical health in mice.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2016.06.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Mgat5 modulates the effect of early life stress on adult behavior and physical health in mice Laura Feldcamp1,2, Jean-Sebastien Doucet2, Judy Pawling3, Marc P Fadel4,5, Paul J Fletcher2, Robert Maunder3,5, James W Dennis3, 6, Albert HC Wong1,2,5,7* 1

Institute of Medical Science, University of Toronto, Medical Sciences Building, 1 King’s College Circle, Room 2374, Toronto, Ontario, Canada, M5S 1A8 2 Campbell Family Mental Health Research Institute, Center for Addiction and Mental Health, 250 College Street, Toronto, Ontario, Canada, M5T 1R8 3 Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario, Canada, M5G 1X5 4 Ontario Shores Centre for Mental Health Sciences, 700 Gordon St, Whitby, Ontario, Canada, L1N 5S9 5 Department of Psychiatry, University of Toronto, 250 College Street, 8th Floor, Toronto, Ontario, Canada, M5T 1R8 6 Department of Molecular Genetics, University of Toronto, Medical Sciences Building, Room 4386, 1 King's College Circle, Toronto, Ontario, Canada, M5S 1A8 and the Department of Laboratory Medicine and Pathology, University of Toronto, Medical Sciences Building, 1 King's College Circle, 6th Floor, Toronto, Ontario, Canada, M5S 1A8 7 Department of Pharmacology, University of Toronto, Medical Sciences Building, Rm 4207, 1 King's College Circle, Toronto, Ontario, Canada, M5S 1A8 *Corresponding author Albert HC Wong Centre for Addiction and Mental Health 250 College St., Room 323 Toronto, Ontario, Canada M5T1R8 Tel: 416-535-8501 x34010 Fax: 416-979-4663

Abbreviated title: Mgat5 mediates early life stress effects in mice

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Highlights - Mgat5-/- mice show a less depression-like phenotype - Mgat5-/- mice have higher bone density and lower blood glucose levels - Cortical neuron spine density is increased in Mgat5-/- males, decreased in females

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Abstract: Psychosocial adversity in early life increases the likelihood of mental and physical illness, but the underlying mechanisms are poorly understood. Mgat5 is an Nacetylglucosaminyltransferase in the Golgi pathway that remodels the N-glycans of glycoproteins at the cell surface. Mice lacking Mgat5 display conditional phenotypes in behaviour, immunity, metabolism, aging and cancer susceptibility. Here we investigated potential gene-environment interactions between Mgat5 and early life adversity on behaviour and physiological measures of physical health. Mgat5-/- mutant and Mgat5+/+ wild-type C57Bl/6 littermates were subject to maternal separation or foster rearing as an early life stressor, in comparison to control mice reared normally. We found an interaction between Mgat5 genotype and maternal rearing condition in which Mgat5-/mice subjected to early life stress had lower glucose levels and higher bone density. Mgat5-/- genotype was also associated with less immobility in the forced swim test and greater sucrose consumption, consistent with a less depression-like phenotype. Cortical neuron dendrite spine density and branching was altered by Mgat5 deletion as well. In general, Mgat5 genotype affects both behaviour and physical outcomes in response to early life stress, suggesting some shared pathways for both in this model. These results provide a starting point for studying the mechanisms by which protein N-glycosylation mediates the effects of early life adversity.

Keywords Mgat5, early life adversity, corticosterone, bone density, neuron spine density

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1.1 Introduction Adverse early life psychosocial experiences have a major impact on later mental health, stress response and physical health [1-3]. In particular, adverse childhood experiences increase risk for mood and anxiety disorders [4], hypothalamic-pituitary axis dysregulation [5], metabolic abnormalities, inflammation, and hypertension [6, 7]. Genetic and epigenetic factors can modify the effect of early psychosocial environment on health [8], and psychological well-being can influence physical health through changes in gene expression [9]. However, much remains to be learned about vulnerability vs. resilience to adversity and whether the effects of early life adversity on physical outcomes are independent of effects on psychological or behavioural parameters [10]. To investigate these questions, we used the Mgat5-/- (mannosyl (alpha-1,6-)glycoprotein beta-1,6-N-acetyl-glucosaminyltransferase 5) mutant mouse. Mgat5-/- mice are resistant to obesity, cancer [11, 12], and depression-related behaviours such as decreased mobility in the forced swim test (FST) and tail suspension test (TST), both at baseline and after chronic mild stress [13]. In animal models, early-life stress results in more anxiety, anhedonia, decreased social interest and social subordinance [4, 14] similar to features of depression or post-traumatic stress disorder [15]. Maternal separation (MS) is a common method for disrupting the neonatal environment [16], that alters the stress response, increases anxiety-related behaviors [17], and impairs pre-pulse inhibition [18]. The resilience of Mgat5-/- mice provides an opportunity to investigate how genetic variation can modify the effect of early life environment on a variety of physiological and behavioral measures in adult animals. The Golgi branching N-acetylglucosaminyltransferases (encoded by Mgat1, Mgat2, Mgat4a/b/c and Mgat5) emerged with vertebrate evolution as an enzymaticallyordered pathway. Cytokine receptors, nutrient transporters and extracellular matrix glycoproteins are modified by the branching pathway, and the Mgat5 modification plays a role in cytokine receptor signaling [19], cell adhesion [20] and migration in the extracellular matrix [21, 22]. Metabolic and epigenetic regulation of the N-glycans on receptor and extracellular matrix may have medium to long-term adaptive effects on cellular responses to environmental conditions [23]. We sought to determine the effect of MS and foster-mother care (FM) on Mgat5-/and Mgat5+/+ mice, in comparison to normal rearing (NR). Both foster rearing and maternal separation represent a disruption of the normal neonatal rearing environment. We measured behaviours relevant to depression in adult mice: tail suspension test (TST), forced swim test (FST) and sucrose preference test (SPT). We also measured physical phenotypes including growth, glucose levels, bone density, corticosterone release under stress, and brain histology. We hypothesized that Mgat5-/- genotype would be protective in a number of domains, suggesting that both psychological and physical resilience can be mediated by the same genetic variation.

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2.1 Materials and Methods 2.1.1 Mice: All procedures were approved by the local Animal Care Committee at the Toronto Centre for Phenogenomics, Toronto, Canada, following guidelines by the University of Toronto and the Canadian Committee for Animal Care. Care was taken to minimize the amount of stress and discomfort mice were subjected to during these experiments. Mice were group housed in environmental isolation with 40 air changes per minute at 21±1°C and 50-60% humidity, with a 12hr light-dark cycle from 0700 to 1900h as mice were housed in a facility shared between groups. Mice had free access to food and water, except for fasting for glucose tolerance testing or the sucrose consumption test. All behavioral experiments were conducted between 0900 to 1700h during the light phase. Cohorts of Mgat5-/- and wild-type (+/+) littermate controls were used in all experiments. Data were pooled for male and female mice when there were no statistically significant differences between the sexes. Mgat5-/- mice were generated as previously described [11] (RRID:MGI_MGI:3578630), and have been backcrossed >20 generations on a C57BL/6J background. Mgat5+/- male mice were mated with female Mgat5+/+, Mgat5+/-, or Mgat5-/mice. Females were housed with sires until plugging was seen (day 1 of pregnancy), and then for a further 14 days after the plugging date. A total of 38 dams generated one litter of pups each. At post-natal day 1 (PND1) pups were randomly assigned to NR (dams and pups were not disturbed), MS (dams were removed from home cage 3hours per day for the first 14 postnatal days), or FM (birth dam was exchanged with a different postpartum Mgat5+/+ female). In all conditions, there was a weekly move to a clean cage. Pups were moved gently and nesting material was transferred to the new cage to reduce cage change stress. In the FM paradigm, mice of all genotypes were raised by a wildtype dam to make the foster experience more uniform, regardless of birth dam genotype. In MS, pups were kept together after dams were removed. Mice were weaned at 26 days and 3-5 mice of the same sex were housed in each cage. Male and female mice of both genotypes were tested at 12-15 weeks of age in the TST, FST, and at 9 to 14 months in SPT, before individual corticosterone sampling. Glucose tolerance (GT) was assessed at 6 months of age, and bone mineral densitometry (BMD) at 8 months. For HPA-axis assessment, blood samples from the saphenous vein of unstressed and stressed animals were taken for comparison of serum corticosterone levels (CORT) between 10 and 14 months. At 14 months brain tissue was collected for Golgi-Cox staining (described below). All mice bred for this project underwent growth measurement and behavioural phenotyping. Randomly selected mice were tested for the remaining physical measurements (BMD, neuron morphology, glucose tolerance, corticosterone). Mice were handled after weaning while being weighed three times per week for five weeks, and all mice underwent the weighing paradigm by the same experimenter in the same manner. 2.1.2 Phenotyping: All mice were phenotyped in the same way, and the tests are described below in the order in which they were conducted. Because all mice were subjected to all tests, an attempt was made to proceed from the least to most stressful tests, in order to minimize 5

the potential stress effects of earlier tests. Other tests like blood glucose and bone density are relevant to older mice, so those were conducted at the appropriate age. Thus, some physical tests such as corticosterone release were conducted later than the related behavioral tests of tail suspension, and forced swim. 2.1.2.1 Growth: Weight was measured 3 times each week until 8 weeks of age to assess post-weaning growth, at PND240, at 14 months, and just before being sacrificed. Equipment was cleaned between mice. 2.1.2.2 TST (tail suspension test): Mice were suspended by the tail from a hook in a sound-attenuated, clear-fronted cabinet and observed for 5 minutes with the Noldus Observer (v5.0.25, Noldus Information Technology, Netherlands). Mice undertook this test 3 times, every other day. The TST and FST (below) were conducted as previously described [13, 24]. Equipment was cleaned between mice. 2.1.2.3 FST (forced swim test): Mice were placed individually into a pyrex glass beaker 17cm diameter x 25cm deep filled ¾ full of water at 22-24°C for 6 minutes. Their movements were recorded and analyzed using Noldus Observer. Water in the beaker was changed between each test. Mice undertook this test twice, two days apart. 2.1.2.4 GT (glucose tolerance): At PND180, 6 male and 6 female mice from each genotype x environment (GxE) group had free-feeding weight and blood glucose measured by obtaining a drop of blood from a tail nick using Bayer Contour® glucometers with Contour® blood glucose test strips (7091C). Mice were fasted 21hr with free access to water, then weighed and fasting blood glucose obtained. Mice were then given an i.p. injection of glucose solution (1.5mg/g anhydrous Dextrose in 0.9% saline, from EMD; DX1256-1; VWR Canada) and blood glucose was measured at 10, 20, 30, 60, 90, and 120 minutes post-injection. 2.1.2.5 BMD (bone mineral density): At PND240, 6 male and 6 female mice from four GxE groups (Mgat5-/- x MS, Mgat5-/- x NR, Mgat+/+ x MS and Mgat5+/+ x NR) underwent x-ray densitometry with the PIXImus Small Animal Densitometer (Piximus, Fitchburg, WI; GE Healthcare Bio-Sciences, Piscataway, NJ). Mice were briefly anesthetized with isoflurane, and imaged with low energy x-rays which were digitally processed. Bone mineral density (BMD), bone mineral content (BMC), lean and fat tissues were analyzed using Lunar PIXImus software for the left femur. Equipment was cleaned between mice. 2.1.2.6 SPT (sucrose preference test): Mice were placed singly in shoebox-style housing for 6 days with free access to food, with one bottle of weighed 1.5% sucrose solution (SUC507.2, Bioshop Canada Inc.) in sterile water, and one bottle of weighed sterile water. Bottles were measured once daily for consumption. 2.1.2.7 Corticosterone: Blood was collected by saphenous vein puncture between the hours of 1200-1400h. 300ul of blood was collected per sampling and mice were allowed to recover for 2 weeks between samplings. Unstressed mice were restrained <5 min in a 50ml Falcon tube for blood collection; stressed mice were gently restrained in a decapicone (DC M200 from Braintree Scientific, Braintree MA, USA) for 30 min before sampling. Blood was collected in EDTA-coated capillary tubes (Microvette CB300 K2 EDTA, Starstedt) then centrifuged for 5 min at 3900g. Serum was separated and stored at -20°C. Corticosterone extraction from serum and corticosterone-specific ELISAs were conducted as per kit instructions (Neogen Corp., KY, USA, cat#402810). Samples were run in triplicate. 6

2.1.2.8 Statistical Analysis: Most data were analyzed with 1, 2 or 3-way ANOVA (sex, rearing environment, genotype) and Tukey’s HSD post-hoc correction for multiple testing. Further analysis employed the Student’s t-test to compare between specific groups. Weight gain data were analyzed with a linear mixed model using Type III Tests of Fixed Effects, and the REML method was used for model fitting [25]. The linear mixed model was: weight = rearing condition (RC) + sex + genotype + day + RC*sex + RC*genotype + RC*day + sex*genotype + sex*day + genotype*day + RC*sex*genotype + RC*sex*day + RC*genotype*day + sex*genotype*day + RC*sex*genotype*day to assess fixed and random effects in the experiment. All data were analyzed in SPSS (15.0.1 or 17) and graphs plotted using GraphPad Prism (v5.02, GraphPad Software Inc.). 2.1.3 Neuron morphology and spine density analysis Mice were anaesthetized with tribromoethanol (Avertin, T48402, Sigma-Aldrich, Canada) perfused with saline, and the brain was removed and bisected down the midline. One half of the brain was placed in Golgi-Cox solution in the dark for 14 days before transfer to 30% sucrose solution for 6 days [26]. 200µm sections were made with a Leica VT1000s microtome and mounted on 2% gelatinized microscope slides. Slides were kept in a humidified chamber for 3 days before fixation and staining. Golgi-cox staining and methods were performed according to standard protocols as previously described [27]. The identity of samples was blinded and only revealed after the analysis was complete. For analysis, pyramidal neurons from the primary and secondary motor cortex at ~0.5 to ~-0.1 from bregma were chosen. Pyramidal neuron spine density and arborization were compared between GxE and sex groups. Images of Golgi stained neurons were taken at 40× magnification under brightfield illumination for dendritic analysis, and at 100x magnification for spine density counts with a Nikon Eclipse E600 microscope. Neurons for analysis were selected based on being fully visible in the 40× magnification field and characterized by clear, distinct morphology. Only pyramidal neurons in layers III and V of the frontal cortex were used, as per a reference atlas [28]. Z-stacks of different focal lengths were generated for each neuron. Neurites were traced using Neuromantic (http://www.rdg.ac.uk/neuromantic); length and surface area, as well as soma body surface area, were recorded. Measurements were normalized to soma surface area. The semi-log Sholl analysis method was used to assess neuron dendrite morphology. ImageJ was used to overlay 11 concentric circles centered at the cell body, each separated by 22μm. The number of times dendrites crossed each circle was counted and the log of this number plotted against the circle radius. The slope of the regression line is defined as κ, the Sholl regression coefficient [29]. The Schoenen ramification index provides a quantitative measure of dendritic branching, and is calculated as the maximum number of intersections divided by the number of primary dendrites [30]. Dendritic spines were counted on the apical dendrites of pyramidal neurons in cortical layers III and V. Spine density was expressed as the number of spines per micrometer of dendrite. All images for quantification were blinded before analysis, and all parameters were normalized to cell soma size, which is correlated with dendritic structure [31]. Differences between GxE groups and sexes were assessed with one-way or twoway ANOVA (SPSS 15.0.1), and Bonferroni correction for multiple testing. Additional 7

confirmation was obtained by Student's two-tailed t test. Data are expressed as mean ± SEM and a significance level of p < 0.05 was used for all analyses.

3.1 Results 3.1.1 Reproductive output: Pregnancy rates were equivalent for both Mgat5-/-, Mgat5+/- and wild-type +/+ Mgat5 mice. Mgat5+/+ dams produced on average 4.0 males (range 3-7) and 2.8 females (range 1-5) per litter, Mgat5+/- dams produced on average 3.8 males per litter (range 2-7 per litter) and 3.6 females per litter (range 2-8 per litter), and Mgat5-/- dams produced on average 3.2 males per litter (range 1-7 per litter) and 3.8 females per litter (range 2-7 per litter). Litter sizes for Mgat5+/+ dams averaged 7.3 pups per litter with a range of 4-10. Litter sizes for Mgat5+/- were average 7.0 pups per litter with a range of 210. Litter sizes for Mgat5-/- dams were 5.9 per litter with a range of 3-10. A total of 38 dams generated 268 pups that survived to weaning and through testing. 3.1.2 Growth: A linear mixed model analysis of weight gain in the first month of life showed significant fixed effects of maternal rearing condition (p=0.004, F 5.56, df 2, 1932000.01) genotype (p<0.0001, F 38.23, df 1, 193200.01), and sex (p<0.0001, F 25.32, df 1, 193200.01) on weight gain. The interaction between genotype and rearing condition also had a significant effect on weight (p=0.001, F 6.51, df 2, 193200.01). With normal rearing, male mutant mice were heavier than wild-type, while female mice of both genotypes were similar in weight (Figure 1a). After maternal separation, male wild type mice were heavier than mutant mice, and this was reversed in females (Figure 1b). With foster rearing, both male and female wildtype mice were heavier than mutants (Figure 1c). Male mice were consistently larger and gained more weight than female mice in the month following weaning, and this was seen in all genotype and rearing groups. Rearing condition had more of an effect on weight gain in mutant mice than in wild-type mice (Figure 1d and e). At 14 months of age, there was considerably more variability in weight between genotypes and rearing conditions than in early life. ANOVA showed female mouse weight to be significantly affected by rearing condition (F7.621 (2,59) p=0.001) and genotype (F75.256(1,59) p<0.001). Tukey’s HSD post-hoc analysis revealed significant differences between female Mgat5-/- and Mgat5+/+ mice (p<0.001). The most striking differences were higher weight in female wild-type mice subjected to maternal separation compared to the other rearing conditions, with female Mgat5-/- mice reared by foster mothers having the lowest weight (Figure 2a). N=8-19 female mice per group. Male weight is significantly affected by genotype only (F9.835(1,39) p=0.003) with post-hoc analysis (Tukey’s HSD) showing differences between MS Mgat5-/- and FM Mgat5+/+ groups (p=0.025). N=6-12 males per group. Male wild-type foster-reared mice had higher weight than Mgat5-/- mice, and Mgat5-/- maternally-separated and foster-reared mice had the lowest weights (Figure 2b). 3.1.3 Behaviour: 8

We assessed performance in three behavioral tests relevant to depression: the tail suspension test (TST), the forced swim test (FST), and preference for sucrose. In the TST, there was a significant effect of Mgat5 genotype on immobility time (ANOVA: F15.296 (1,114) p<0.001), with Mgat5-/- mice moving more overall. There was also a significant genotype x rearing condition interaction effect on TST immobility time (ANOVA: F4.153 (1,114) p=0.002), but no clear effect of rearing environment. In the Tukey HSD post-hoc analysis, normally-reared Mgat5-/- mice did not differ from Mgat5+/+ wild-type littermates, but foster-rearing resulted in significant lower TST inactivity in Mgat5-/- mice compared to foster-reared or normally-reared Mgat5+/+ mice (Figure 3a). There was no impact of sex on time inactive in TST (F1.474 (1,114) p=0.119), nor for environment (F1.587(2,114) p=0.209). As with the FST, the TST has been proposed as a screening test for antidepressants, with more movement typically interpreted as predictive of antidepressant effects [32]. As reported previously [13], there was a strong effect of Mgat5 genotype on the FST in both trials, with Mgat5-/- mice swimming more than wild type controls, regardless of rearing condition, which did not seem to affect FST performance (Figure 3b). Repeated testing of the FST resulted in more immobility in Mgat5-/- mice, with a lesser effect in wild-type mice. Two-tailed paired t-tests were conducted to assess differences between trial 1 and trial 2 for each genotype x environment group. There was no significant difference in the percent change in immobility time between trials, when comparing Mgat5-/- and Mgat5+/+ mice. One-way ANOVA for trial 1: F13.405(5,120), p<0.001. Tukey HSD post hoc p<0.005 between Mgat5-/- and Mgat5+/+ groups and environmental rearing conditions. For trial 2: F9.345(5,120), p<0.001. Tukey HSD post hoc p<0.03 between Mgat5-/- and Mgat5+/+ groups and environmental rearing conditions. N=14-28 mice per group. Large effects of genotype (F18.768(1,114) p<0.001) and sex (F39.808(1,114) p<0.001) on sucrose consumption were seen, with an interaction between genotype and sex (F4.135(1,114) p=0.044). No clear effect of rearing environment on sucrose consumption was seen. Sex-specific analysis showed genotype effects in both males (F7.276(1,44) p=0.01) and females (F6.001(1,70) p<0.001). Post hoc analyses showed a significant difference between genotypes for male FM mice, and for MS and FM females. There were no differences between genotypes in sucrose intake when mice were reared normally, but foster rearing resulted in significantly less sucrose consumption in wildtype mice relative to Mgat5-/- for females (p=0.004). Male mice raised by foster mothers also showed decreased sucrose consumption in the wild-type group vs. Mgat5-/- mice (p=0.011: Figure 3c). 3.1.4 Neuron morphology: There was no clear pattern of changes in dendritic spine density based on rearing environment or genotype. However, there were more subtle differences when certain subgroups were compared. Significant genotype x environment group differences in spine density were seen for both sexes together (F13.346(3,172) p<0.001). Post hoc analysis showed significant differences in spine density between most groups of male mice. A gene x environment group by sex effect was seen (one-way ANOVA: F21.633(3,327) p<0.001). In male mice, Mgat5 deficiency increased spine density regardless of rearing condition (Figure 4a). In female mice, the opposite effect was 9

observed: Mgat5-/- genotype was associated with lower spine density after maternal separation (ANOVA F13.346(3,172) p<0.001). (Figure 4b). Sholl analysis is used to quantify dendritic branching complexity, with a higher regression coefficient indicating more branching [29]. There was a significant effect of sex (F19.616(1,111) p<0.001) and genotype x environment group in the semi-log Sholl analysis (F6.986(2,111) p<0.001) without a significant interaction between the two. Mgat5 -/- mice generally had more complex dendritic trees than wild-type mice (p=0.016 comparing genotypes in both MS and NR groups). However, this genotype effect appears to be driven by differences in male mice, where normally-reared Mgat5-/- had higher Sholl numbers (p=0.027 N = 36-50 neurons per group) than normally-reared wild type males (Figure 4c). In female mice, there were no significant differences between groups., . 3.1.5 Glucose tolerance: Glucose levels were measured in 6 month-old free-feeding mice and in mice injected with glucose in solution after 21 hours of fasting, because this is relevant to diabetes and metabolic health. Overall, most Mgat5-/- mice had lower glucose levels after early-life stress. There was a significant effect of sex and genotype on glucose levels (fasted: sex, F21.693(1,60),p<0.001; genotype, F6.640(1,60), p=0.012 and freefeeding: sex, F17.249(1,60), p<0.001; genotype, F6.667(1,60), p=0.012). In male Mgat5-/mice, maternal separation and foster rearing resulted in lower fasting glucose levels than in wild-type mice (Student’s two-tailed t-test p=0.045 and p=0.013, respectively: Figure 5a and 5b). Similarly, foster-reared female Mgat5-/- mice had lower free-feeding glucose levels than their wild-type controls (Figure 5c). Male Mgat5-/- mice raised in FM conditions had better glucose tolerance than NR mice (Figure 5d). Wild-type mice did not show significant differences in glucose tolerance when different rearing conditions were compared (data not shown). 3.1.6 Bone mineral density: The most interesting result was that maternal separation during early life decreased bone density in adult Mgat5+/+ but not Mgat5-/- mice. Overall, neonatal rearing condition had a significant effect on bone mineral density (one-way ANOVA: F4.667 (1,45) p=0.036). Because there was an interaction of sex x genotype x environment (F5.492(1,41) p=0.024) we analyzed males and females separately. For male mice, twoway ANOVA (environment x genotype), indicated significant differences between male MS Mgat5+/+ vs. NR Mgat5+/+ groups (F4.722(1,20), p=0.0420). The Bonferroni post hoc test indicated significant differences in bone mineral density between MS Mgat5+/+ vs. NR Mgat5+/+ (p=0.0383), as well as between MS Mgat5-/- vs. MS Mgat5+/+ (two-tailed Student’s t-test, p=0.0402; N=6-7 mice per group) (Figure 6a). Female mice showed no significant differences in bone density between rearing conditions or genotype groups (Figure 6b). 3.1.7 Corticosterone: The knockout mice appeared to have more stress reactivity than wild-type mice as measured by change in corticosterone levels after restraint stress. All mice were briefly restrained (<5min) for unstressed samples, and after 3 weeks, were restrained for 30 min before blood sampling. There was a significant sex effect (ANOVA F11.219(1,60) 10

p=0.001) on corticosterone levels (ANOVA F49.693(1,60) p<0.001). In female mice, there was a significant GxE interaction effect on corticosterone release after restraint stress (ANOVA F4.234 (5,30) p=0.005). Post hoc testing indicated that female MS Mgat5-/- mice had significantly higher corticosterone release than all wild-type females (Figure 7). FM Mgat5-/- unstressed female mice showed a trend towards an effect of rearing environment (ANOVA F2.891(2,3) p=0.072). In male mice, there was an overall effect of genotype on corticosterone release after stress (ANOVA F4.427 (1,30) p=0.044), but there were no significant differences in post hoc testing between individual groups divided by genotype or rearing condition. 4.1 Discussion We found that the Mgat5 gene interacts with neonatal rearing disruptions to affect a variety of phenotypes at different stages of development and aging. Disruption of the early-life early life environment in Mgat5-/- mice reduced dendritic spine density and increased corticosterone levels after restraint stress, compared to wild-type mice. On the other hand, Mgat5-/- mice exposed to early life stress had better glucose tolerance and higher bone density. Mgat5-/- mice also had less depression-related behaviors than wildtype mice, regardless of rearing conditions. Our findings support the notion of complex gene-environment interactions influencing susceptibility to common disease [33]. We also observed effects on phenotypes in a variety of organ systems that are often considered unrelated in the clinical domain, highlighting the potential overlap in pathophysiology among diseases traditionally considered distinct, such as depression and metabolic disorders. In particular, the TST, FST and sucrose consumption tests are relevant to depression, while glucose levels and bone density are relevant for diabetes and osteoporosis, respectively. The relative resilience of Mgat5-/- mice on behavioral measures relevant to depression and in bone density and glucose metabolism suggests that this gene and biochemical pathway of N-glycan remodelling may be important for mediating the effects of early life adversity. 4.1.1 TST, FST and sucrose consumption as measures of depressive behaviour These three behavioral tests were chosen because of their relevance to human depression. The FST was originally conceived as a test for antidepressant effects from medication, and most antidepressants increase swimming time in this test. Some have also suggested that immobility in the FST is a marker of “behavioral despair” in rodents, which could be related to feelings of hopelessness in depression [34]. The genetic background of mice also affects performance in the FST [35]. Sucrose intake has been proposed as an animal behavioral indicator of a depression-like state, but it has also been used to assess antidepressant effects [36]. Overall, we did not observe a consistent effect of rearing environment on these behaviour tests. However, Mgat5-/- genotype had effects on behaviour that are similar to that of antidepressant medications, especially in female mice and sucrose consumption. These results are consistent with previous reports [13] and suggest that Mgat5 deficiency could be protective for depression. 4.1.2 Growth and weight maintenance in adulthood 11

The decreased weight of Mgat5-/- mice compared to wild type mice, both during development and as adults is consistent with previous reports [13]. Not surprisingly, male mice were heavier than female mice at the same time point and within the same genotype or rearing condition. In Mgat5-/- mice, disruption to maternal care slowed weight gain but this effect was minimal in wild-type mice. In late life (14 months), Mgat5-/- females weighed significantly less than wild type females, regardless of rearing condition. However, wild type females exposed to maternal separation weighed more. This is consistent with the finding of increased obesity in adult humans with a history of maltreatment in childhood [37, 38]. For males, there was not as strong an effect of genotype. Normally reared Mgat5-/- male mice were the same weight as their wild type counterparts. Only maternally separated Mgat5-/- males showed a significant difference in weight when compared to wild type males reared by foster mothers. 4.1.3 Glucose tolerance Glucose metabolism measurements showed a general trend for significantly decreased resting blood glucose levels in Mgat5-/- male mice versus wild type mice in maternally separated and foster mothered groups. In females there was significantly greater blood glucose after glucose challenge in the foster mother group for wild type mice. In male Mgat5-/- mice, disrupted rearing decreased blood glucose levels and improved glucose tolerance, in contrast to the increased risk of diabetes in people who experience childhood adversity [39]. The Mgat5-modified glycans on the glucagon receptor promote sensitivity to glucagon, and therefore Mgat5-/- mice produce less hepatic glucose and display faster glucose clearance [40], which appears to be further enhanced by disrupted maternal care. These results suggest that Mgat5 gene deletion is protective against a diabetes-like phenotype by promoting better glucose metabolism. 4.1.4 Bone mineral density Male Mgat5-/- maternally separated mice have significantly greater bone density than wild type maternally separated mice. These data suggests that the absence of Mgat5 protects against the deleterious effects of early-life adversity on bone mineral density. This contradicts previous reports in which the absence of Mgat5 has been shown to produce autoimmune hypersensitivity, resistance to weight gain, osteoporosis, decreased muscle mass and satellite cell renewal [40], as well as an increased risk of autoimmunity and demyelination [41]. Although not directly pertinent to bone density, Mgat5 SNPs are associated with severity of multiple sclerosis in humans [42], consistent with increased sensitivity to autoimmune encephalomyelitis in Mgat5-/- mice [43]. Glycosylation has a role in pathogenesis and the regulation of the immune system, for example in rheumatoid arthritis and diabetes [44] and glycosylation of proteins is important in the pathogenesis of autoimmune disease [45]. 4.1.5 Mgat5 modulates neurodevelopment We did not directly examine potential molecular mechanisms that could link Mgat5 deletion with disrupted neonatal rearing to affect these diverse outcomes. However, there is evidence that Mgat5 is involved in neuronal development and metabolism that could account for our observations. The N-glycans processed by Mgat5 are needed for normal development of neural tissue through TrKA interactions with 12

Nerve Growth Factor (NGF), which promotes neuronal survival and differentiation, and regulates aspects of neurite outgrowth such as cell polarity, motility and the formation of cell processes [46]. Our analysis of neuron dendrite structure and spine density shows modest changes as a result of both Mgat5 genotype and maternal separation, in the frontal cerebral cortex. However, the findings for male mice and female mice were in the opposite direction, and thus not entirely consistent with the behavioral and glucocorticoid measurements. Studying other brain regions more directly involved in mood regulation and stress response might provide better insight into the structural basis for the behavioral and hormonal observations. 4.1.6 Early life adversity affects hypothalamic-pituitary-adrenal (HPA) axis function and brain Stress early in life such as maternal separation in rats and adverse rearing conditions in nonhuman primates produces long-lived hyperactivity of glucocorticoid systems, and greater reactivity of the HPA axis in response to adult stress [5]. Epigenetic mechanisms, specifically altered methylation of the glucocorticoid receptor gene promoter, represent one pathway transducing early life environmental inputs into adult stress response and behavioral set-points [14]. Dysfunctional HPA axis regulation in response to external stress and hormonal changes is seen in a number of psychiatric conditions including depression and post-traumatic stress disorder (PTSD) [4]. The prefrontal cortex, amygdala, hippocampus, and HPA axis are particularly sensitive to early-life adversity, which can produce lasting molecular and cellular changes. These include changes in synaptic structure and density, NMDA-receptor mediated signaling, attenuated neurogenesis and long-term potentiation deficits [47]. Even a short exposure to maternal separation can increase plasma corticosterone levels [48] and change hippocampal structure [49], further supporting a neurodevelopmental basis for behavioral abnormalities induced by early-life adversity. Our corticosterone results show a somewhat irregular pattern, with some indication that Mgat deletion is associated with more corticosterone release in response to stress. Thus, there may be other mechanisms affecting the behavioral changes we observed, other than the HPA axis, or perhaps further investigation of other HPA parameters would be helpful. The C57Bl/6J mouse used in this experiment carries a spontaneous homozygous deletion in the nicotinamide nucleotide transhydrogenase (Nnt) gene (GenBank: AH009385.2) [50]. The product of this gene can directly affect the function of steroidogenic mitochondria and adrenal response to stress within the HPA axis. This feature could account for the irregular pattern of response to stress or in corticosterone levels [51]. 4.1.7 Sex differences We found several significant differences in outcomes between male and female mice, which suggest that the sex of the animal can modulate gene-environment interactions. In particular, female Mgat5-/- female mice exposed to maternal separation gained less weight gain than wild-type controls, while the opposite genotype effect was seen in male mice (Fig 1b). Mgat5-/- mice generally consumed more sucrose than wildtype mice, but this effect was greater in female mice that had a disrupted neonatal rearing 13

environment. Perhaps the most striking sex differences were seen with neuronal dendrite spine density. In male mice, the Mgat5-/- genotype group had more spines, with maternal separation appearing to further increase spine number. Conversely, in female mice, Mgat5-/- the genotype group had fewer spines, and maternal separation was also associated with fewer spines (Figs 4a and 4b). Mgat5-/- genotype was protective against the negative effects of maternal separation on bone loss, but only in male mice (Fig 6). This specific finding is of potential clinical relevance since age-related osteoporosis is more often a concern with women than men. Finally, female mice showed a stronger gene-environment interaction on their corticosterone response to restraint stress than male mice. Taken together, these results indicate that sex is an important variable in modulating gene-environment interactions between Mgat5 and the neonatal environment, and it would be interesting to investigate the mechanisms underlying these sex differences. 4.1.8 Vulnerability and resilience An important question for understanding the relationship between stressors and disease is why some individuals are sensitive, while others are more resilient and resistant to the effects of these stressors [10]. A well-known example is PTSD, which typically affects only a subset of individuals exposed to trauma. Even when a group is exposed to essentially the same external traumatic event, which can occur in military operations, only some soldiers suffer from PTSD [52]. A recent study found that polymorphic variation in the FKBP5 gene alters chromatin interactions that influence stress hormone release and susceptibility to PTSD [53]. Of special relevance to our study is that the clinical effect of this FKBP5 susceptibility variant was dependent on childhood trauma, providing an elegant example of the complex interplay between genetic, epigenetic and environmental factors in the pathophysiology of adult psychiatric disorders. Our results show that Mgat5 affects susceptibility to the effects of early life stress, and this provides some insight into mechanisms influencing resilience. There is also evidence that some behavioral phenotypes confer resilience; for example, high noveltyseeking rats are more resilient against maternal separation, and do not exhibit the typical negative behavioral and neuroendocrine consequences [54]. Others have found that epigenetic mechanisms mediate the variable effects of maternal separation on mice of different genetic backgrounds [55]. The different results observed after MS and FM in both Mgat5-/- and wild-type mice suggests that the type of stressor can affect whether the animal is seen as vulnerable or resilient. The two early life disruptions were chosen because they are well-established and commonly studied, but they are of course artificial experimental interventions with limited direct translatability to real-world stressors. However, the results do highlight that susceptibility and resilience can be different in the context of closely-related stressors. However, not all genetic mutations interact with maternal separation to affect behaviour. For example, deletion of the serotonin 1A receptor does not [56], demonstrating the importance of identifying the specific gene-environment interactions that affect resilience. Interestingly, Mgat5-/- first-time mothers housed alone displayed less time gathering pups and nurturing whereas two Mgat5-/- mothers housed together were more efficient at rearing pups as measured by survival [40]. Evolutionary pressures to maximize litter size may strain parental resources and thus create early-life stress [57, 14

58]. In accordance with a hormesis model, a relatively low dose of early-life stresses may be optimal for survival, whereas excessive distress above a threshold leads to loss of resilience and pathology in adult life. The effect of toxic stress in childhood on adult health is so strong that some have argued for many adult diseases to be conceptualized as developmental disorders beginning in early life [59]. Depression is a good example of a disorder influenced by early life environment [1], and it is also associated with lower bone mineral density and osteoporosis [60-62]. In our study we found that Mgat5-/- mice were relatively resistant to depression-related behaviours regardless of early environment. When maternal care was disrupted, Mgat5-/- mice were resistant to loss of bone density in males, and had lower glucose levels in most conditions. However, the changes in dendritic spine density and corticosterone levels did not match the behavioral and physical phenotype outcomes. Another important question is whether the behavioral and physical outcomes we measured are independent or not. It is easy to rationalize that some physical outcomes such as increased bone density or lower adult weight could be secondary to a behavioral pattern such as increased activity. Because there was no clear effect of early life adversity on behaviour in the Mgat5-/- mice, it is not possible to make direct conclusions about the independence of these types of outcomes from our experiment. This is a limitation of our study design, and it would be useful to study other animal models or to apply other experimental interventions that could reproduce some of the outcomes while sparing others. This could be the focus of future research efforts. Another limitation is that our study did not identify a specific mechanism linking the disruption in early life or genetic mutation with the outcomes. Knowledge of molecular mechanisms could permit more targeted manipulations that could help to determine whether the behavioral and physical outcomes are separate or arise from the same core pathways. 5.1 Conclusion In conclusion, we have found a new role for Mgat5 in modifying susceptibility to both physical and behavioral effects of early life stress. The protective effects of Mgat5-/genotype in depression-related behaviour, bone density and glucose metabolism suggest that they may share overlapping origins. This knowledge adds to our understanding of how gene-environment interactions influence health, and provides a starting point for further work to identify particularly vulnerable individuals or groups. Future experiments could also focus on developing interventions that may attenuate the negative effects of disrupted early development. Acknowledgements This paper was funded through internal funds at the Centre for Addiction and Mental Health (CAMH), which had no bearing on interpretation or analysis of the results.

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References [1] Caspi A, Sugden K, Moffitt TE, Taylor A, Craig IW, Harrington H, et al. Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene. Science. 2003;301:386-9. [2] Nelson CA, 3rd, Zeanah CH, Fox NA, Marshall PJ, Smyke AT, Guthrie D. Cognitive recovery in socially deprived young children: the Bucharest Early Intervention Project. Science. 2007;318:1937-40. [3] Miller GE, Chen E, Parker KJ. Psychological stress in childhood and susceptibility to the chronic diseases of aging: moving toward a model of behavioral and biological mechanisms. Psychol Bull. 2011;137:959-97. [4] Penza KM, Heim C, Nemeroff CB. Neurobiological effects of childhood abuse: implications for the pathophysiology of depression and anxiety. Arch Womens Ment Health. 2003;6:15-22. [5] Plotsky PM, Meaney MJ. Early, postnatal experience alters hypothalamic corticotropin-releasing factor (CRF) mRNA, median eminence CRF content and stress-induced release in adult rats. Brain Res Mol Brain Res. 1993;18:195-200. [6] Danese A, Moffitt TE, Harrington H, Milne BJ, Polanczyk G, Pariante CM, et al. Adverse childhood experiences and adult risk factors for age-related disease: depression, inflammation, and clustering of metabolic risk markers. Arch Pediatr Adolesc Med. 2009;163:1135-43. [7] Stein DJ, Scott K, Haro Abad JM, Aguilar-Gaxiola S, Alonso J, Angermeyer M, et al. Early childhood adversity and later hypertension: data from the World Mental Health Survey. Ann Clin Psychiatry. 2010;22:19-28. [8] Hertzman C, Boyce T. How experience gets under the skin to create gradients in developmental health. Annu Rev Public Health. 2010;31:329-47 3p following 47. [9] Fredrickson BL, Grewen KM, Coffey KA, Algoe SB, Firestine AM, Arevalo JM, et al. A functional genomic perspective on human well-being. Proc Natl Acad Sci U S A. 2013;110:13684-9. [10] Rutter M. Implications of resilience concepts for scientific understanding. Ann N Y Acad Sci. 2006;1094:1-12. [11] Granovsky M, Fata J, Pawling J, Muller WJ, Khokha R, Dennis JW. Suppression of tumor growth and metastasis in Mgat5-deficient mice. Nat Med. 2000;6:306-12. [12] Cheung P, Dennis JW. Mgat5 and Pten interact to regulate cell growth and polarity. Glycobiology. 2007;17:767-73. [13] Soleimani L, Roder JC, Dennis JW, Lipina T. Beta Nacetylglucosaminyltransferase V (Mgat5) deficiency reduces the depression-like phenotype in mice. Genes Brain Behav. 2008;7:334-43. [14] Weaver IC, Cervoni N, Champagne FA, D'Alessio AC, Sharma S, Seckl JR, et al. Epigenetic programming by maternal behavior. Nat Neurosci. 2004;7:847-54. [15] Heim C, Newport DJ, Bonsall R, Miller AH, Nemeroff CB. Altered pituitaryadrenal axis responses to provocative challenge tests in adult survivors of childhood abuse. Am J Psychiatry. 2001;158:575-81. [16] Newport DJ, Stowe ZN, Nemeroff CB. Parental depression: animal models of an adverse life event. Am J Psychiatry. 2002;159:1265-83. 16

[17] Huot RL, Plotsky PM, Lenox RH, McNamara RK. Neonatal maternal separation reduces hippocampal mossy fiber density in adult Long Evans rats. Brain Res. 2002;950:52-63. [18] Ellenbroek BA, Cools AR. Early maternal deprivation and prepulse inhibition: the role of the postdeprivation environment. Pharmacol Biochem Behav. 2002;73:177-84. [19] Lau KS, Partridge EA, Grigorian A, Silvescu CI, Reinhold VN, Demetriou M, et al. Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation. Cell. 2007;129:123-34. [20] Partridge EA, Le Roy C, Di Guglielmo GM, Pawling J, Cheung P, Granovsky M, et al. Regulation of cytokine receptors by Golgi N-glycan processing and endocytosis. Science. 2004;306:120-4. [21] Lagana A, Goetz JG, Cheung P, Raz A, Dennis JW, Nabi IR. Galectin binding to Mgat5-modified N-glycans regulates fibronectin matrix remodeling in tumor cells. Mol Cell Biol. 2006;26:3181-93. [22] Goetz JG. Bidirectional control of the inner dynamics of focal adhesions promotes cell migration. Cell Adh Migr. 2009;3:185-90. [23] Dennis JW, Nabi IR, Demetriou M. Metabolism, cell surface organization, and disease. Cell. 2009;139:1229-41. [24] Lipina TV, Zai C, Hlousek D, Roder JC, Wong AH. Maternal immune activation during gestation interacts with Disc1 point mutation to exacerbate schizophreniarelated behaviors in mice. J Neurosci. 2013;33:7654-66. [25] Searle SR, Casella G, McCulloch CE. Variance Components. Hoboken, New Jersey: John Wiley & Sons, Inc.; 2006. [26] Gibb R, Kolb B. A method for vibratome sectioning of Golgi-Cox stained whole rat brain. J Neurosci Methods. 1998;79:1-4. [27] Lee FH, Fadel MP, Preston-Maher K, Cordes SP, Clapcote SJ, Price DJ, et al. Disc1 point mutations in mice affect development of the cerebral cortex. J Neurosci. 2011;31:3197-206. [28] Paxinos G, Watson C. The mouse brain in stereotaxic coordinates. 3rd ed. San Diego, CA: Academic Press Inc.; 2012. [29] Sholl DA. Dendritic organization in the neurons of the visual and motor cortices of the cat. J Anat. 1953;87:387-406. [30] Schoenen J. Dendritic organization of the human spinal cord: the motoneurons. J Comp Neurol. 1982;211:226-47. [31] Somogyi P, Klausberger T. Defined types of cortical interneurone structure space and spike timing in the hippocampus. J Physiol. 2005;562:9-26. [32] Steru L, Chermat R, Thierry B, Simon P. The tail suspension test: a new method for screening antidepressants in mice. Psychopharmacology (Berl). 1985;85:367-70. [33] Gibson G. Decanalization and the origin of complex disease. Nat Rev Genet. 2009;10:134-40. [34] Porsolt RD, Bertin A, Jalfre M. Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther. 1977;229:327-36. [35] Porsolt RD, Bertin A, Jalfre M. "Behavioural despair" in rats and mice: strain differences and the effects of imipramine. Eur J Pharmacol. 1978;51:291-4. 17

[36] Willner P, Towell A, Sampson D, Sophokleous S, Muscat R. Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant. Psychopharmacology (Berl). 1987;93:358-64. [37] Van Niel C, Pachter LM, Wade R, Jr., Felitti VJ, Stein MT. Adverse events in children: predictors of adult physical and mental conditions. J Dev Behav Pediatr. 2014;35:549-51. [38] Hemmingsson E, Johansson K, Reynisdottir S. Effects of childhood abuse on adult obesity: a systematic review and meta-analysis. Obes Rev. 2014;15:882-93. [39] Gilbert LK, Breiding MJ, Merrick MT, Thompson WW, Ford DC, Dhingra SS, et al. Childhood Adversity and Adult Chronic Disease: An Update from Ten States and the District of Columbia, 2010. Am J Prev Med. 2014. [40] Cheung P, Pawling J, Partridge EA, Sukhu B, Grynpas M, Dennis JW. Metabolic homeostasis and tissue renewal are dependent on beta1,6GlcNAc-branched Nglycans. Glycobiology. 2007;17:828-37. [41] Lee SU, Grigorian A, Pawling J, Chen IJ, Gao G, Mozaffar T, et al. N-glycan processing deficiency promotes spontaneous inflammatory demyelination and neurodegeneration. J Biol Chem. 2007;282:33725-34. [42] Brynedal B, Wojcik J, Esposito F, Debailleul V, Yaouanq J, Martinelli-Boneschi F, et al. MGAT5 alters the severity of multiple sclerosis. J Neuroimmunol. 2010;220:120-4. [43] Demetriou M, Granovsky M, Quaggin S, Dennis JW. Negative regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation. Nature. 2001;409:733-9. [44] Descamps FJ, Van den Steen PE, Nelissen I, Van Damme J, Opdenakker G. Remnant epitopes generate autoimmunity: from rheumatoid arthritis and multiple sclerosis to diabetes. Adv Exp Med Biol. 2003;535:69-77. [45] Opdenakker G, Dillen C, Fiten P, Martens E, Van Aelst I, Van den Steen PE, et al. Remnant epitopes, autoimmunity and glycosylation. Biochim Biophys Acta. 2006;1760:610-5. [46] Yang X, Li J, Geng M. N-acetylglucosaminyltransferase V modifies TrKA protein, regulates the receptor function. Cell Mol Neurobiol. 2008;28:663-70. [47] Roth TL, Sweatt JD. Epigenetic marking of the BDNF gene by early-life adverse experiences. Horm Behav. 2010. [48] De la Fuente M, Llorente R, Baeza I, De Castro NM, Arranz L, Cruces J, et al. Early maternal deprivation in rats: a proposed animal model for the study of developmental neuroimmunoendocrine interactions. Ann N Y Acad Sci. 2009;1153:176-83. [49] Llorente R, Llorente-Berzal A, Petrosino S, Marco EM, Guaza C, Prada C, et al. Gender-dependent cellular and biochemical effects of maternal deprivation on the hippocampus of neonatal rats: a possible role for the endocannabinoid system. Dev Neurobiol. 2008;68:1334-47. [50] Kraev A. Parallel universes of Black Six biology. Biol Direct. 2014;9:18. [51] Goldstein DS. Adrenal responses to stress. Cell Mol Neurobiol. 2010;30:143340.

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[52] Iversen AC, Fear NT, Simonoff E, Hull L, Horn O, Greenberg N, et al. Influence of childhood adversity on health among male UK military personnel. Br J Psychiatry. 2007;191:506-11. [53] Klengel T, Mehta D, Anacker C, Rex-Haffner M, Pruessner JC, Pariante CM, et al. Allele-specific FKBP5 DNA demethylation mediates gene-childhood trauma interactions. Nat Neurosci. 2013;16:33-41. [54] Clinton SM, Watson SJ, Akil H. High novelty-seeking rats are resilient to negative physiological effects of the early life stress. Stress. 2014;17:97-107. [55] Kember RL, Dempster EL, Lee TH, Schalkwyk LC, Mill J, Fernandes C. Maternal separation is associated with strain-specific responses to stress and epigenetic alterations to Nr3c1, Avp, and Nr4a1 in mouse. Brain Behav. 2012;2:455-67. [56] Groenink L, Bijlsma EY, van Bogaert MJ, Oosting RS, Olivier B. Serotonin1A receptor deletion does not interact with maternal separation-induced increases in startle reactivity and prepulse inhibition deficits. Psychopharmacology (Berl). 2011;214:353-65. [57] Hager R, Johnstone RA. The genetic basis of family conflict resolution in mice. Nature. 2003;421:533-5. [58] Stockley P, Parker GA. Life history consequences of mammal sibling rivalry. Proc Natl Acad Sci U S A. 2002;99:12932-7. [59] Shonkoff JP, Garner AS. The lifelong effects of early childhood adversity and toxic stress. Pediatrics. 2012;129:e232-46. [60] Charles LE, Fekedulegn D, Miller DB, Wactawski-Wende J, Violanti JM, Andrew ME, et al. Depressive symptoms and bone mineral density among police officers in a northeastern US City. Glob J Health Sci. 2012;4:39-50. [61] Cizza G, Mistry S, Nguyen VT, Eskandari F, Martinez P, Torvik S, et al. Do premenopausal women with major depression have low bone mineral density? A 36-month prospective study. PLoS One. 2012;7:e40894. [62] Diem SJ, Harrison SL, Haney E, Cauley JA, Stone KL, Orwoll E, et al. Depressive symptoms and rates of bone loss at the hip in older men. Osteoporos Int. 2013;24:111-9.

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Figure Legends Figure 1. Effects of Mgat5 and rearing condition on body weight from 3 days post weaning to 28 days post weaning. A) Mgat5 NR mice (-/- and +/+ genotypes). B) MS mice (-/- and +/+ genotypes) weight gain after weaning. C) FM mice (-/- and +/+ genotypes) weight gain after weaning. D) Mgat5+/+ mice across all three environmental rearing conditions E) Mgat5-/- mice weight gain after weaning for all three rearing conditions. Figure 2. Body weight at 14 months of age in both female and male mice. A) Female mouse weight was significantly affected by rearing condition and genotype. Tukey’s HSD post-hoc ***p<0.001, **p<0.015, *p<0.042; NR (normal rearing); N=8-19 female mice per group. B) Male weight is significantly affected by genotype only (ANOVA F9.835(1,39) p=0.003) with post-hoc analysis (Tukey’s HSD) showing differences between MS Mgat5-/- and FM Mgat5+/+ groups (*p=0.025) (#p=0.055). N=6-12 male mice per group. Figure 3. Mgat5-/- mice show less depression-related behaviors. A) Tail suspension test (TST) results for both male and female mice. Mgat5 genotype was the main driver of differences between groups (ANOVA F15.296(1,114) p<0.001). Post hoc Tukey HSD ** p<0.008, # p=0.076. B) Forced swim test (FST) is affected by Mgat5 genotype and rearing condition. N=14-28 mice per group. MS Mgat5-/- (n=18) *p=0.008; FM Mgat5-/- (n=14) **p=0.013; NR NR Mgat5-/- (n=16) ***p<0.001; MS Mgat5+/+ (n=27) **p=0.011; FM Mgat5+/+ (n=28) p=0.142; and NR Mgat5+/+ (n=23) p=0.227. Both male and female mice are shown together. C) Sucrose consumption is affected by Mgat5 genotype, sex and rearing condition, with an interaction between genotype and sex. N=5-22 per group (female); n=6-17 per group (male). # p=0.087 **p=0.004 * p=0.011. Figure 4. Dendritic spine density and branching is affected by Mgat5 and rearing condition. A) Male Mgat5-/- mice have higher frontal cortical pyramidal neuron spine density (Tukey’s HSD: NR Mgat5-/- vs. NR Mgat5+/+ **p=0.01, NR Mgat5-/- vs. MS Mgat5-/*p=0.048, MS Mgat5-/- vs. MS Mgat5+/+ ***p=0.007, NR Mgat5+/+ vs. MS Mgat5-/***p<0.001). B) Female Mgat5-/- mice have lower frontal cortical pyramidal neuron spine density (Tukey’s HSD: NR-/- vs NR+/+ **p=0.01, NR-/- vs MS-/- *p=0.048, MS-/- vs MS+/+ ***p=0.007, NR+/+ vs MS-/- ***p<0.001). Females: n = 28-59 neurons per group. C) Sholl regression coefficient analysis of dendritic branching is affected by genotype (**p=0.027). Males: N = 36-50 neurons per group. Figure 5. Mgat5-/- mice have lower glucose levels. 20

A) Mgat5-/- genotype is associated with lower fasted glucose levels in male mice exposed to maternal separation (*p=0.045, Student’s two-tailed t-test). B) Mgat5-/- genotype is associated with lower fasted and free-feeding glucose levels in male mice raised by foster mothers (**p=0.013, Student’s two-tailed t-test). C) Mgat5-/- genotype is associated with lower free-feeding glucose levels in female mice raised by foster mothers (*p=0.048, Student’s two-tailed t-test). D) Foster-reared male Mgat5-/- mice had faster glucose normalization after glucose challenge than NR mice (one-way ANOVA: t=0 (F3.099(2,15), p=0.075, post hoc Tukey’s HSD: FM Mgat5-/- vs. NR Mgat5-/- p=0.074); t=90min (F3.046(2,15), p=0.078, post hoc p=0.068); t=120min (F4.435(2,15), p=0.031, post hoc p=0.03). N=6 per group Figure 6. Bone Mineral Density. A) Maternal separation decreased bone density in male Mgat5+/+ mice but not Mgat5-/mice. (*p=0.0383, **p=0.0402 MS Mgat5+/+ vs both MS and NR Mgat5-/-; N=6-7 mice per group) B) Female groups had no significant differences in bone mineral density. Figure 7. Corticosterone levels in female mice before and after stressor challenge. Female stressed mice showed a significant effect of genotype x environment (F4.234(5,30) p=0.005), post-hoc tests indicate differences between MS Mgat5-/- and all Mgat5+/+ groups (**p=0.015, ***p=0.008, #p=0.089).

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