Wnt/BDNF pathway mediates autism-like behaviors induced by prenatal subclinical hypothyroidism in male neonatal rats

Wnt/BDNF pathway mediates autism-like behaviors induced by prenatal subclinical hypothyroidism in male neonatal rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Wnt/BDNF pathway mediates autism-like behaviors induced by prenatal subclinical hypothyroidism in male neonatal rats DIJIE LIU, Kai Tao, Ying Sun, Jialin Hao, Shiyong Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4675145/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Translational Psychiatry → Version 1 posted 11 You are reading this latest preprint version Abstract Epidemiological studies have illuminated that abnormal maternal thyroid function in early pregnancy escalates the risk of autism spectrum disorder (ASD) in offspring by nearly fourfold. However, the exact mechanism by which maternal thyroid dysfunction affects the risk of ASD in the offspring remains unknown. Early pregnancy emerges as a pivotal juncture for fetal brain development, with the fetus heavily reliant on maternal thyroid hormones for its neurological maturation. Our investigation unveils a striking correlation: mothers afflicted by perinatal subclinical hypothyroidism (SCH) tend to have male offspring displaying marked ASD-like behavioral anomalies, typified by conspicuous diminution in social interactions and repetitive behavioral patterns. Furthermore, we discerned a substantial reduction in neuron count within critical brain regions among offspring of SCH mothers, suggestive of underlying neurodegeneration or developmental impediments. Moreover, diminished levels of brain-derived neurotrophic factor (BDNF), cAMP response element binding protein (CREB), and Bcl-2 were noted in the hippocampal region of SCH offspring, juxtaposed with a noteworthy upregulation of mTOR expression and downregulation of Wnt. These findings coalesce to suggest that the Wnt-mediated signaling pathway, in concert with its interplay with the downstream target BDNF, might underpin one of the mechanisms by which maternal SCH predisposes offspring to autism-like behaviors. Health sciences/Diseases Biological sciences/Genetics Subclinical hypothyroidism hippocampus ASD offspring Rat Figures Figure 1 INTRODUCTION Autism spectrum disorder (ASD) presents as a complex neurodevelopmental condition characterized by social impairments, repetitive behaviors, and restricted interests( 1 ). Over recent years, the prevalence of ASD has surged, attributed partly to enhanced diagnostic criteria and increased medical awareness ( 2 , 3 ). According to data from the Centers for Disease Control and Prevention (CDC), the prevalence of ASD in children has risen from approximately 1 in 44 in 2018 to 1 in 36 in 2023 ( 4 ). However, the exact mechanisms driving this escalation remain unknown( 5 ). Additionally, emerging research suggests a robust association between maternal thyroid function during pregnancy and the risk of psychiatric disorders in offspring, including attention deficit hyperactivity disorder (ADHD), schizophrenia, and ASD ( 6 , 7 ). A meta-analysis by Levie et al. demonstrated a 1.8-fold increase in autism risk among offspring born to mothers with isolated hypothyroxinemia (IH) ( 8 ). Moreover, a study in the Netherlands uncovered a nearly fourfold heightened risk of ASD in offspring at age 6 years when pregnant women experienced severe IH in early pregnancy ( 9 ). Given the pivotal role of thyroid hormones in brain development( 10 ), particularly during the critical period of early pregnancy when the fetal hypothalamic-pituitary-thyroid (HPT) axis remains immature, maternal thyroid dysfunction can profoundly impact neurodevelopment ( 9 , 11 ). In this study, we probe the potential link between maternal SCH and neurodevelopmental disorders, with a specific focus on ASD. Employing a highly standardized approach mirroring autism-specific behaviors in humans, we comprehensively assess the impact of maternal SCH during pregnancy on offspring neurodevelopment. Our findings reveal that maternal SCH during pregnancy and lactation induces ASD-like behavioral abnormalities and diminishes the number of hippocampal neurons in offspring. Furthermore, we observe decreased expression of pivotal molecules involved in hippocampal neurogenesis and synaptic plasticity, namely BDNF, CREB, and Bcl-2. Additionally, we explore the gene expression profiles of m-TOR and Wnt, given their established roles in neural development and synaptic function. In conclusion, our study, utilizing a rat model of SCH during pregnancy, sheds light on autism-like behavioral alterations in offspring, accompanied by hippocampal developmental deficits. We further elucidate the effects on the Wnt signaling pathway and the CREB-BDNF pathway, aiming to contribute to a deeper understanding of this critical area of research and provide guidance for future investigations. MATERIALS AND METHODS Animals Specific pathogen-free nulliparous female Wistar rats (n = 24), weighing 180-200g, were used in all experimental procedures. They were housed in a climate-controlled specific-pathogen-free laboratory (temperature 25°C ± 1°C and relative humidity 55%) with a 12-hour light/dark photoperiod. All animals were permitted free access to normal rat chow and tap water. All experimental procedures were approved by Animal Care and Use Committee at China Medical University, which complies with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Thyroidectomy surgery and drug delivery A total of 24 female rats were randomized into control and SCH groups, with 12 rats in each group. According to our previous study ( 12 ), all surgeries were performed after intraperitoneal injection of 3% sodium pentobarbital (0.1 ml/100 g). Briefly, the anesthetized rats were fixed on the operating table, and the skin was incised along the midline of the neck, and the subcutaneous tissue and sternocleidomastoid muscle were bluntly separated. The thyroid glands were located on either side of the trachea. A midline incision was made in the isthmus, and the thyroid gland was carefully separated from the trachea with forceps while avoiding injury to the recurrent laryngeal nerve, and then penicillin was gradually injected after suturing to prevent infection. After surgery, to prevent possible hypocalcemia due to destruction of the parathyroid glands by total thyroidectomy, rats were provided with 0.1% (w/v) calcium lactate in the drinking water postoperatively and fed with normal rat chow. One month after the rats were operated, L-T 4 (Sigma, USA) was continuously infused as a daily subcutaneous injection of 1.0-1.05 µg/100g. All female rats were mated with normal male rats (female to male ratio = 2:1). The following day, when spermatozoa were detected on microscopic vaginal smears, this day was designated as day 0 (E0) of pregnancy. Pregnant females were housed in individual cages until delivery, and the day of birth of the offspring was designated as postnatal day 0 (P0). Blood (approximately 2 ml) was drawn from all groups of female rats on days E7, E13, and P1 after injection to monitor total T 4 and TSH levels. The schedule of the project is shown in (Fig. 1). Serum hormone levels assessments Blood samples obtained from rats were immediately centrifuged at 13,000 rpm for 15 min and stored at -80°C. TT 4 was calculated by Roche electrochemiluminescence (Roche Diagnostic Products, Los Angeles, CA, USA). TSH was determined by ELISA kit for thyroid-stimulating hormone (Cloud-Clone Corp., Houston, TX, USA). The inter- and intra-assay coefficients of variation (CVs) for TT 4 were 3.38–4.26% and 1.37–1.79%, respectively. The inter-assay and intra-assay coefficients of variation (CVs) for thyroid-stimulating hormone (TSH) were < 10% and < 12%, respectively. All samples were duplicated. Behavioral tests Self-grooming The behavioral procedure was as described previously ( 13 ). The rats were individually placed in a clean, standard open space (40 cm×40 cm) and the experiment lasted for a total of 20 min, with the first 10 min being the acclimatization phase, during which the total time spent grooming and the total number of times the rats groomed during the second 10 min were measured. The duration of the rat's self-grooming behavior was also recorded and videotaped with a timer at a location 1.5 m away from the cage. Self-grooming was defined as licking paws, licking legs, washing nose and face, or scratching head, body, fur and tail with any paw. Three-chambered Social Test A total of five-week-old rats were used to determine sociality and preference for social novelty. The test consisted of three 10-min phases, including habituation, sociability, and novelty preference. Before each phase, the test mouse was maintained in the middle chamber while two doorways were closed. In the first phase, the test mouse was allowed to habituate in the three-chamber arena for 10 min. In the second sociability phase, the test mouse was exposed to an age-matched unfamiliar male mouse (stranger 1) confined in a wire cage, which was located in one of the chambers, and an identical empty wire cage was placed at the corresponding spot in the opposite chamber. In the third novelty preference phase, an age and sex-matched novel mouse (stranger 2) was enclosed in the empty wire cage, and the test mouse was allowed to roam between chambers containing familiar mouse (stranger 1) and novel mouse (stranger 2). All parameters, including the time spent in each chamber and the sniffing time with the unfamiliar rats (Stranger 1 and Stranger 2), were recorded and calculated using the automated Noldus Observer software (Ethovision 11.0). Open-field test (OFT) Open-field locomotion in a novel environment was assessed as previously described( 13 ). The open-field apparatus was made of gray plywood with dimensions of 40 × 40 cm and a wall height of 30cm. Rats were placed in the center of the open field and their movements were recorded with a video camera fixed to the top of the apparatus for 10 or 30 min and analyzed with Ethovision 11.0 (Noldus). The test apparatus was cleaned with 70% ethanol between two subjects and then wiped clean with a clean paper towel. The center area of the empty field instrument was 25% of the total area (approximately 25 cm × 25 cm quare). Morris Water Maze Test (MWM) The MWM test was performed as previously described( 12 ). Before the test, rats were allowed to swim freely in a pool without a platform for 60 seconds. For spatial learning, the rats were trained to find the underwater platform within 60 seconds on each trial. If the rats failed to find the hidden platform within 60 seconds, they were manually guided to the platform and stayed there for 30 seconds. The training was conducted 3 times a day for 4 days. On day 5 for the spatial memory test, the platform was removed and escape latency, number of platform traversals, duration in the platform quadrant, and average swimming speed were monitored using Noldus Observer software (Ethovision 11.0). Tissue Processing and Histopathology Staining The histomorphology of the hippocampus was observed using H&E and Nissl staining at P44. Five to eight young rats from each group were anesthetized with 10% chloral hydrate and then transcardially perfused with cold saline and 4% paraformaldehyde (PFA) in 0.01M phosphate-buffered saline (PBS, pH 7.4) for 30 minutes. The brains were subsequently removed and stored in 4% PFA in PBS at 4°C overnight, followed by cryoprotection in 30% sucrose at 4°C. Coronal sections were embedded in paraffin, and consecutive 5-µm-thick coronal sections of the frontal lobes were cut and stained with H&E and toluidine blue (Nissl staining method). The total integrated optical density (IOD), representing the level of Nissl bodies in the CA1 region of the hippocampus, was measured at 40× and 400× magnification using a cast-grid microscope (MetaMorph/DP10/Bx41, UIC/OLYMPUS, US/JP) and an image analysis program (MetaMorph offline 4.65). Western Blotting Hippocampal samples from P44 rats were collected and processed as described in our previous study. Proteomes were extracted using a BCA kit [Pierce™, Rockford, IL, USA], and then 0.2% bromophenol blue and 7.5% β-mercaptoethanol were added and mixed by pipetting. The proteome extracts were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in TBS-T (50 mM Tris HCl, pH 7.4, 100 mM NaCl, 0.2% Tween-20) for 30 minutes with gentle stirring. The membranes were then incubated overnight at 4°C with primary antibodies: BDNF (1:1,000; Cell Signaling Technology), CREB (1:1,000; Cell Signaling Technology), Bcl-2 (1:1,000; Cell Signaling Technology), and β-actin (1:1,000; Santa Cruz Biotechnology). Following incubation, the membranes were rinsed twice with distilled water, then rinsed with TBS-T, and subsequently incubated for 1 hour at room temperature with horseradish peroxidase-conjugated anti-mouse/anti-rabbit IgG (1:10,000–15,000). After additional rinsing with TBS-T, the experiment was repeated five times. The optical density values of the target bands were analyzed using a gel image processing system (Bio-Rad). Quantitative real-time PCR Total RNA was obtained from hippocampal tissue using TRIzol reagent (Thermo Fisher Scientific, USA) according to the manufacturer's instructions and its concentration was determined using a microplate reader (Molecular Devices, USA). Total RNA was then reverse transcribed to cDNA using the Revert Aid First Strand cDNA Synthesis Kit (Fermentas, USA) following the manufacturer's instructions. using the Quantitect SYBR Green PCR Kit (Thermo Fisher Scientific, USA) q-PCR was performed on an ABI PRISM 7500 Real-Time System (Applied Biosystems, USA). The amplification parameters were as follows: denaturation at 95°C for 30 seconds, annealing at 95°C for 5 seconds, extension at 60°C for 30 seconds, 40 cycles, and detection of signal at 60°C. Data were quantified using the 2 −ΔΔCt method and normalized to β-actin expression. Primer sequences (Sangon Biotech) were: m-TOR 5'-ATCGTGCTGTTGGGTGAGAG-3' and 5'-TGGATC TCCAGCTCTCCGAA-3'; Wnt1, 5'-AACAGTAGTGGCCGATGGTG-3' and 5'- GGGTTCTGTCGGGATCAGTCG-3'; β-actin, 5'-GGCTGTATTCCCCTCCATCG-3' and 5'-CCAGTTGGTAACAATG CCATGT-3'. Statistical analysis All data are expressed as mean ± standard error of mean (SEM). All statistical analyses were performed using SPSS software. Comparisons were analyzed using one-way ANOVA followed by Tukey's test. One-way repeated ANOVA was used for the escape latency data of MWM. All graphs were analyzed using Graph Pad Prism 8 (GraphPad Software Inc.). Statistical significance was defined at * P < 0.05, ** P < 0.01. RESULTS Thyroid Hormone Levels in Maternal Rats To assess the success of maternal SCH model, we measured serum TT4 and TSH levels on days 7 and 13 of pregnancy and on the day of delivery. The results showed that TSH levels were significantly higher in the SCH group compared to the control group (on all days tested; P < 0.05), but TT 4 levels were not statistically different from the control group. The results met the criteria for SCH and confirmed the successful establishment of the maternal SCH rat model. The mean body weights of the littermates at P40 were as follows: control group, 122.11 ± 3.66 g; SCH group, 117 ± 4.75 g. There was no significant difference(Table 1, Fig .2). SCH offspring rats show increased stereotyped behaviors We evaluated the stereotypic behaviors of offspring rats by recording the frequency and duration of behaviors like licking paws, washing nose and face, or scratching fur with feet during their free-range activities (Fig. 3A). The results showed that the frequency and duration of self-grooming stereotypic behaviors increased significantly in male rats in the SCH group compared to control rats ( P < 0.01), while the total distance traveled during these activities did not change significantly (Fig. 3B). In the female group, there was a statistically significant increase in the frequency of stereotypic behaviors in the SCH group compared to the control group ( P < 0.05), although no significant difference was observed in the duration and total distance traveled during the activity (Fig. 3C). These findings indicate that SCH offspring rats exhibited more frequent grooming behaviors, which are suggestive of typical autistic behaviors. Social interaction deficits in SCH offspring rats The social activity behaviors of the two groups of offspring rats were examined using a three-chambered social test (Fig. 4A, B). Our study found that male rats in the control group spent significantly more time sniffing Stranger Rat 1 than the empty cage ( P < 0.01), indicating that the control group males did not have significant social behavioral deficits. However, male rats in the SCH group showed no significant difference in sniffing time between Stranger Rat 1 and the empty cage, suggesting that they exhibited significant social behavioral deficits. Similarly, in the social novelty preference and motivation tests, control group males spent significantly more time sniffing Stranger Rat 2 than Stranger Rat 1 ( P < 0.01), demonstrating normal social novelty preference behavior. In contrast, male rats in the SCH group did not show a significant difference in sniffing time between Stranger Rat 1 and Stranger Rat 2, indicating ASD-like behaviors such as novelty preference deficits and decreased socialization (Fig. 4C). In the female group, there was no significant difference between the SCH group and the control group in the time spent sniffing Stranger 1 and the empty cage. In the social novelty preference test, only the control group females showed normal social novelty preference behavior, spending significantly more time sniffing Stranger Rat 2 than Stranger Rat 1( P < 0.01). However, female rats in the SCH group showed no significant difference in sniffing time between Stranger Rat 1 and Stranger Rat 2, indicating a significant social novelty preference deficit (Fig. 4D). Open-field test (OFT) In order to exclude the possibility that locomotor dysfunction may lead to social deficits, we assessed motor performance in the open field test (Fig. 5A), in which a statistically significant difference was observed in the SCH male group compared to the control group in terms of the time spent in the central area of the open field ( P < 0.05), suggesting the presence of anxious behaviours in the SCH male offspring rats (Fig. 5B). In both female groups, no significant difference was observed in the time spent in the central area of the open field in the SCH group compared to the control group. In addition, there was no significant difference in the total distance travelled between the male and female groups. (Fig. 5C). Spatial learning and memory impairment in SCH offspring rats Autism is often accompanied by intellectual disabilities and memory impairment, so we conducted Morris Water Maze (MWM) tests to assess these aspects. The rats were divided into cages based on sex, and the experiment was conducted exclusively on male offspring rats (Fig. 6A). On the first day of the spatial learning experiment, the rats were in the acclimatization period, so there was no significant difference in the mean escape latency (i.e., the time taken to reach the hidden platform) between the groups. From the second day of training, the mean escape latency of the rats in each group decreased as the number of training sessions increased. However, from the second to the fifth day, the SCH group showed a slower decrease in escape latency and spent significantly more time searching for the hidden platform compared to the control group. ANOVA indicated that this difference was statistically significant ( P < 0.01, Fig. 6B). On the last day, during the probe trials phase, the SCH group spent significantly less time in the target quadrant compared to the control group, with the difference being statistically significant ( P < 0.01, Fig. 6C). Additionally, the frequency of crossing the platform area was significantly lower in the SCH group, and this difference was also statistically significant ( P < 0.05, Fig. 6D). However, there was no significant difference in the average swimming speed between the SCH group and the control group, suggesting that the SCH group exhibited impaired spatial learning and memory (Fig. 6E). Damage of SCH to neurons in the hippocampal CA1 region of offspring rats Red arrows point to representative Nissl bodies. The IOD values of Nissl bodies in the hippocampal CA1 region of SCH male offspring rats were significantly reduced compared to those of the control group. The Nissl bodies in the SCH group were either broken and missing, or the neurons were irregularly arranged and poorly delaminated. These results suggest that maternal SCH causes dissolution and loss of Nissl bodies in hippocampal neurons of male offspring, disrupting neuronal structure and function (Fig. 7A, B). Compared to the normal control, statistically significant differences were observed in the IOD of Nissl bodies in the CA1 region of the hippocampus in the male group ( P < 0.01, Fig. 7C), while no significant difference was seen in the IOD of the female group (Fig. 7D). Effect of SCH on hippocampal CREB, BDNF, and Bcl-2 levels in offspring rats We observed that hippocampal CREB levels were significantly decreased in male offspring rats in the SCH group compared to the control group ( P < 0.01). Additionally, we investigated whether BDNF, a downstream molecule of CREB, mediated the effects of maternal SCH on autism-like behaviors in offspring rats. The results revealed that hippocampal BDNF levels were significantly lower in male offspring rats of the SCH group compared to the control group ( P < 0.01). Furthermore, Bcl-2 expression was also significantly decreased in the SCH male group, with the difference being statistically significant ( P < 0.05). Similarly, hippocampal CREB and BDNF levels were significantly decreased in female offspring rats of the SCH group compared to the control group ( P < 0.01). However, while Bcl-2 expression was slightly decreased in the female SCH group, the difference was not statistically significant (Fig. 8A, B). Effect of SCH on m-TOR and Wnt gene expression in offspring rats In male offspring rats of the SCH group, the expression of the target gene m-TOR was significantly elevated compared to the control group ( P < 0.01). Conversely, Wnt expression was significantly lower in the SCH male group compared to the control group ( P < 0.01). In female offspring rats, m-TOR expression was slightly elevated in the SCH group compared to the control group, but this difference was not statistically significant. Similarly, Wnt gene expression was slightly decreased in the SCH female group compared to the control group, but again, the difference was not statistically significant (Fig. 9A, B). DISCUSSION This study is the first to directly investigate the molecular mechanisms by which maternal SCH induces autism-like behaviors in offspring. The key findings of this study are as follows: First, SCH during gestation and lactation induces autism-like behaviors in male offspring rats, such as increased grooming behavior and social interaction deficits. Second, the integrated optical density of Nissl staining in the CA1 region of the hippocampus was lower in male offspring of SCH dams compared to controls. Third, maternal SCH led to a significant reduction in hippocampal BDNF, CREB, and Bcl-2 protein levels, a significant up-regulation of m-TOR gene expression, and a significant down-regulation of Wnt gene expression in the offspring compared to sham-operated control groups. Additionally, we observed sex-specific phenotypic differences. In conclusion, this study provides direct evidence that maternal SCH results in motor and/or social interaction impairments in offspring. The Wnt signaling pathway, along with thyroid-regulated genes BDNF, CREB, and Bcl-2, play critical roles in the pathogenesis of autism-like behavioral abnormalities in the offspring of SCH mothers. Over the past decade, increasing evidence has shown that maternal subclinical hypothyroidism (SCH) impairs learning and memory functions in offspring( 11 , 14 ). The hippocampus is a vital brain region in the central nervous system (CNS) involved in regulating emotions and memory( 15 ). It contains a high density of thyroid hormone receptors and various neurotransmitters( 16 ) and serves as a significant regulator of the HPT axis( 17 ). The cornu ammonis (CA) region of the hippocampus, primarily composed of pyramidal cells, is crucial for processing emotional information( 18 ) and forming short-term memory( 19 ). cAMP response element-binding protein (CREB) is a nuclear transcription factor that plays a pivotal role in neuronal development, including neuronal survival, proliferation, synapse formation, synaptic plasticity, and long-term memory formation( 19 , 20 ). CREB-dependent transcription is critical for various forms of learning and memory( 21 ). Previous research has shown that iodine deficiency leads to a reduction in total CREB and phosphorylated CREB (p-CREB) levels in the hippocampus of lactating and adolescent rats( 22 ). In this study, we found a decrease in CREB protein levels in the offspring of maternal SCH. Brain-derived neurotrophic factor (BDNF), a downstream target of CREB( 23 ), is synthesized by neurons and astrocytes and plays a crucial role in the development and survival of neural stem cells (NSCs) by binding to TrkB receptors( 21 ). Our findings demonstrated a significant reduction in hippocampal BDNF protein levels in the offspring of maternal SCH, consistent with previous studies( 12 ). Alterations in BDNF are linked to core psychopathological features of autism, as BDNF is essential for synaptic plasticity and cognitive functions( 24 ). Since CREB acts as a transcriptional activator of BDNF through motifs preceding BDNF exon IV( 25 ), the down-regulation of BDNF proteins in the hippocampus of the offspring may be mediated by reduced CREB activity( 26 ). Impaired CREB-BDNF signaling has been widely implicated in the pathogenesis of autism( 25 ), and our results further support the disruption of this signaling cascade in the context of maternal SCH. Thyroid hormone can directly activate the apoptotic pathways involving the Bcl-2 family, in addition to influencing the brain neurotrophic factor pathway( 27 ). Bcl-2 is crucial for neuronal survival and apoptosis regulation( 28 ). Research by Kim et al. and Jang et al. has demonstrated that thyroid hormone deficiency induces neuronal apoptosis in rat models( 29 ). Dysregulation of anti-apoptotic Bcl-2 family members has been implicated in mood disorders such as anxiety, depression, and bipolar disorder in humans. Moreover, deletion of the Bcl-2 gene in mouse neural progenitor cells has been associated with anxiety-like behavior in rats( 30 ). Our study reveals that maternal SCH significantly reduces Bcl-2 protein expression in the brains of offspring rats. Although we did not perform apoptosis staining, it is plausible that apoptosis could occur and be completed during gestation( 30 ). The complexity of apoptotic processes necessitates further in-depth studies to fully elucidate the role of Bcl-2 dysregulation in the context of maternal SCH and its effects on offspring brain development. mTOR is a crucial effector protein downstream of the PI3K/Akt pathway( 31 ), integrating multiple extracellular signals involved in protein synthesis and synaptic plasticity( 32 ). Brain organoid culture experiments by Takei et al. demonstrated that abnormalities in the PI3K/AKT/mTOR signaling pathway affect cortical neurogenesis and induce autism-like behavioral changes( 33 ). mTOR promotes synaptic plasticity and memory by regulating protein synthesis( 34 ), and mutations in its pathway components are widely associated with neurodevelopmental disorders( 35 ). For instance, peripheral blood mononuclear cells from patients with mild and severe idiopathic autism exhibit heightened mTOR signaling activity, suggesting it as a molecular signature of clinical ASD severity( 36 ). Consistent with previous reports, our study showed that male offspring of the SCH group displayed autism-like behavioral phenotypes, with a significant upregulation of mTOR gene expression in the hippocampus. Notably, the mTOR-dependent protein translation process in the cytoplasmic lysate of neural circuits is regulated by axonal BDNF/TrkB signaling, providing a plausible explanation for our findings( 37 ). Existing literature underscores the critical role of the Wnt pathway in neurodevelopmental disorders( 38 ). Disruption of Wnt signaling during the embryonic development of the cerebral cortex has been linked to delayed neuronal migration and abnormal gyrus connectivity( 39 ), leading to a range of neurodevelopmental disorders such as ASD( 40 ). In the present study, we observed significant downregulation of the Wnt gene in the male offspring of SCH rats, along with behavioral abnormalities such as sensory dysfunction, social deficits, and obsessive-compulsive behaviors. These findings suggest that dysregulation of the Wnt pathway may be one mechanism by which SCH leads to the development of neuropsychiatric disorders in offspring. Additionally, Liu et al. have associated the PI3K-Akt-Wnt pathway with improvements in short-term memory following exercise( 41 ), while Park et al. have elucidated the relationship between BDNF expression and Wnt signaling( 42 ). Wang et al. and Yang et al. have demonstrated the importance of Wnt signaling in neural development and its regulatory relationship with BDNF( 40 , 43 ). These studies emphasize the importance of the Wnt pathway and BDNF signaling in neurodevelopmental disorders, especially considering the autism-like behaviors observed in male rats, consistent with our findings. It is well-documented that the prevalence of ASD is significantly higher in males than in females( 44 , 45 ), indicating that innate biological factors influence the manifestation of neurodevelopmental disorders differently between the sexes( 46 ). Both genetic and hormonal factors contribute to these gender differences. Several studies have identified sex-specific genetic variants associated with ASD, with specific genes on the X and Y chromosomes potentially contributing to the higher male prevalence( 47 ). CONCLUSIONS In conclusion, our study provides evidence that maternal subclinical hypothyroidism (SCH) influences the Wnt pathway and brain-derived neurotrophic factor (BDNF) levels in offspring rats, with particularly pronounced effects in males. These sex-specific findings, including autistic-like behaviors, underscore the potential neurodevelopmental impact of maternal SCH. Declarations ACKNOWLEDGMENTS AND DISCLOSURES This work was supported by The National Natural Science Foundation of China (Grant: 81200573 and 82103338). Liaoning Province Science and Technology Planning Project (Grant 2021JH2/10300108) The authors report no biomedical financial interests or potential conflicts of interest. References Shan ZY, Chen YY, Teng WP, Yu XH, Li CY, Zhou WW, et al. (2009): A study for maternal thyroid hormone deficiency during the first half of pregnancy in China. Eur J Clin Investigation 39: 37–42. Sealey LA, Hughes BW, Sriskanda AN, Guest JR, Gibson AD, Johnson-Williams L, et al. (2016): Environmental factors in the development of autism spectrum disorders. Environment International 88: 288–298. Baron-Cohen S, Auyeung B, Nørgaard-Pedersen B, Hougaard DM, Abdallah MW, Melgaard L, et al. (2015): Elevated fetal steroidogenic activity in autism. Mol Psychiatry 20: 369–376. Lord C, Elsabbagh M, Baird G, Veenstra-Vanderweele J (2018): Autism spectrum disorder. 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Grønli J, Bramham C, Murison R, Kanhema T, Fiske E, Bjorvatn B, et al. (2006): Chronic mild stress inhibits BDNF protein expression and CREB activation in the dentate gyrus but not in the hippocampus proper. Pharmacology Biochemistry and Behavior 85: 842–849. Liu F-G, Hu W-F, Wang J-L, Wang P, Gong Y, Tong L-J, et al. (2017): Z-Guggulsterone Produces Antidepressant-Like Effects in Mice through Activation of the BDNF Signaling Pathway. International Journal of Neuropsychopharmacology 20: 485–497. Ma K, Taylor C, Williamson M, Newton SS, Qin L (2023): Diminished activity-dependent BDNF signaling differentially causes autism-like behavioral deficits in male and female mice. Front Psychiatry 14: 1182472. Seok S, Fu T, Choi S-E, Li Y, Zhu R, Kumar S, et al. (2014): Transcriptional regulation of autophagy by an FXR–CREB axis. Nature 516: 108–111. Guan L, Jia N, Zhao X, Zhang X, Tang G, Yang L, et al. (2013): The involvement of ERK/CREB/Bcl-2 in depression-like behavior in prenatally stressed offspring rats. Brain Res Bull 99: 1–8. Basu (2010): PKCε induces Bcl-2 by activating CREB. Int J Oncol 36. https://doi.org/10.3892/ijo_00000566 Kim SS, Jang SA, Seo SR (2013): CREB‐mediated Bcl‐2 expression contributes to RCAN1 protection from hydrogen peroxide‐induced neuronal death. J of Cellular Biochemistry 114: 1115–1123. Wang P, Xu J, Zhang C (2010): CREB, a possible upstream regulator of Bcl-2 in trichosanthin-induced HeLa cell apoptosis. Mol Biol Rep 37: 1891–1896. Li Y, Zhou Y, Liu D, Wang Z, Qiu J, Zhang J, et al. (2023): Glutathione Peroxidase 3 induced mitochondria-mediated apoptosis via AMPK /ERK1/2 pathway and resisted autophagy-related ferroptosis via AMPK/mTOR pathway in hyperplastic prostate. J Transl Med 21: 575. Hu Y, Mai W, Chen L, Cao K, Zhang B, Zhang Z, et al. (2020): mTOR‐mediated metabolic reprogramming shapes distinct microglia functions in response to lipopolysaccharide and ATP. Glia 68: 1031–1045. Takei N, Furukawa K, Hanyu O, Sone H, Nawa H (2014): A possible link between BDNF and mTOR in control of food intake. Front Psychol 5. https://doi.org/10.3389/fpsyg.2014.01093 Park J, Yoon Y-S, Han H-S, Kim Y-H, Ogawa Y, Park K-G, et al. (2014): SIK2 Is Critical in the Regulation of Lipid Homeostasis and Adipogenesis In Vivo. Diabetes 63: 3659–3673. Ly C, Greb AC, Cameron LP, Wong JM, Barragan EV, Wilson PC, et al. (2018): Psychedelics Promote Structural and Functional Neural Plasticity. Cell Reports 23: 3170–3182. Steinhart Z, Angers S (2018): Wnt signaling in development and tissue homeostasis. Development 145: dev146589. Yi H, Hu J, Qian J, Hackam AS (2012): Expression of brain-derived neurotrophic factor is regulated by the Wnt signaling pathway. NeuroReport 23: 189–194. Moy S, Nadler J, Young N, Perez A, Holloway L, Barbaro R, et al. (2007): Mouse behavioral tasks relevant to autism: Phenotypes of 10 inbred strains. Behavioural Brain Research 176: 4–20. Liu J, Xiao Q, Xiao J, Niu C, Li Y, Zhang X, et al. (2022): Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Sig Transduct Target Ther 7: 3. Wang K, Zhang R, Lehwald N, Tao G-Z, Liu B, Liu B, et al. (2023): Wnt/β-catenin signaling activation promotes lipogenesis in the steatotic liver via physical mTOR interaction. Front Endocrinol 14: 1289004. Liu S, Liu Q, Ju Y, Liu L (2021): Downregulation of miR-383 reduces depression-like behavior through targeting Wnt family member 2 (Wnt2) in rats. Sci Rep 11: 9223. Park S-S, Kim S-H, Kim B-K, Shin M-S, Jeong H-T, Park J-S, Kim T-W (2023): Treadmill exercise ameliorates chemotherapy-induced memory impairment through Wnt/β-catenin signaling pathway. J Exerc Rehabil 19: 314–319. Yang J-W, Ma W, Luo T, Wang D-Y, Lu J-J, Li X-T, et al. (2016): BDNF promotes human neural stem cell growth via GSK-3 β -mediated crosstalk with the wnt/ β -catenin signaling pathway. Growth Factors 34: 19–32. Tiwari A, Khera R, Rahi S, Mehan S, Makeen HA, Khormi YH, et al. (2021): Neuroprotective Effect of α-Mangostin in Ameliorating Propionic Acid-Induced Experimental Model of Autism in Wistar Rats. Brain Sciences 11: 288. Choi GB, Yim YS, Wong H, Kim S, Kim H, Kim SV, et al. (2016): The maternal interleukin-17a pathway in mice promotes autism-like phenotypes in offspring. Science 351: 933–939. Bohmwald K, Andrade CA, Mora VP, Muñoz JT, Ramírez R, Rojas MF, Kalergis AM (2022): Neurotrophin Signaling Impairment by Viral Infections in the Central Nervous System. IJMS 23: 5817. Werling DM, Geschwind DH (2013): Sex differences in autism spectrum disorders: Current Opinion in Neurology 26: 146–153. Table Table 1 is available in the Supplementary Files section Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files Table1.docx Cite Share Download PDF Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Translational Psychiatry → Version 1 posted Editorial decision: revise 04 Dec, 2024 Review # 3 received at journal 19 Oct, 2024 Review # 2 received at journal 05 Oct, 2024 Reviewer # 3 agreed at journal 27 Sep, 2024 Review # 1 received at journal 25 Sep, 2024 Reviewer # 2 agreed at journal 19 Sep, 2024 Reviewer # 1 agreed at journal 18 Sep, 2024 Reviewers invited by journal 18 Sep, 2024 Submission checks completed at journal 03 Jul, 2024 Editor assigned by journal 02 Jul, 2024 First submitted to journal 02 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4675145","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":322165869,"identity":"871c1e09-a208-4217-b4c3-c8f8c96c86da","order_by":0,"name":"DIJIE LIU","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACPmYGBokEHgkGfvYGhgNEaWGDaZHsOUCsFiCWADEMbiQQ6TA2dh7DGw9kLOQYbj5/eLighkGeX4yAZWzMPMYWQIcZM87OMTg84xiD4czZBKwDajED+SWxWTqH4TAPG0OCwW0itdS3SR5/cJjnHwlaQIFmcJi3jSgtbMUgvxjO4AH6hbdPgrBf+PkPb7z5s6dO3v748cefeb7ZyPNLE9ACBow9cKYEEcrB4AexCkfBKBgFo2BEAgCvozcBtLR12wAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0006-0207-8495","institution":"China Medical University","correspondingAuthor":true,"prefix":"","firstName":"DIJIE","middleName":"","lastName":"LIU","suffix":""},{"id":322165870,"identity":"dcd86fbf-d04e-4933-a5c0-309dc8481216","order_by":1,"name":"Kai Tao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Tao","suffix":""},{"id":322165871,"identity":"e40a7848-c4cd-486b-aa9c-90bc56bba7af","order_by":2,"name":"Ying Sun","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Sun","suffix":""},{"id":322165872,"identity":"bb69b76c-b899-4d69-9b26-2dfbb461fc53","order_by":3,"name":"Jialin Hao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jialin","middleName":"","lastName":"Hao","suffix":""},{"id":322165873,"identity":"7a5d8588-8a3e-43fa-9695-89f9ad0d3801","order_by":4,"name":"Shiyong Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shiyong","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-07-02 14:57:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4675145/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4675145/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41398-025-03570-6","type":"published","date":"2025-10-07T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62116527,"identity":"273bd3eb-bafc-4521-9080-48f0923577f6","added_by":"auto","created_at":"2024-08-09 12:56:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":769805,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4675145/v1/822d788c1a793224eb5fe36a.jpg"},{"id":93008908,"identity":"c6f485fc-4ce1-4b2e-b158-6017e481f95f","added_by":"auto","created_at":"2025-10-08 07:05:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1532330,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4675145/v1/f9bef8de-9b12-4fa1-8f92-8e42c543da4a.pdf"},{"id":62115693,"identity":"5cb52e77-f15b-4951-9ffc-51c2b2fc6694","added_by":"auto","created_at":"2024-08-09 12:48:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":34883,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4675145/v1/14dbca196eb27a2cfa0f8777.docx"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Wnt/BDNF pathway mediates autism-like behaviors induced by prenatal subclinical hypothyroidism in male neonatal rats","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAutism spectrum disorder (ASD) presents as a complex neurodevelopmental condition characterized by social impairments, repetitive behaviors, and restricted interests(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Over recent years, the prevalence of ASD has surged, attributed partly to enhanced diagnostic criteria and increased medical awareness (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). According to data from the Centers for Disease Control and Prevention (CDC), the prevalence of ASD in children has risen from approximately 1 in 44 in 2018 to 1 in 36 in 2023 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). However, the exact mechanisms driving this escalation remain unknown(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Additionally, emerging research suggests a robust association between maternal thyroid function during pregnancy and the risk of psychiatric disorders in offspring, including attention deficit hyperactivity disorder (ADHD), schizophrenia, and ASD (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). A meta-analysis by Levie et al. demonstrated a 1.8-fold increase in autism risk among offspring born to mothers with isolated hypothyroxinemia (IH) (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Moreover, a study in the Netherlands uncovered a nearly fourfold heightened risk of ASD in offspring at age 6 years when pregnant women experienced severe IH in early pregnancy (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Given the pivotal role of thyroid hormones in brain development(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), particularly during the critical period of early pregnancy when the fetal hypothalamic-pituitary-thyroid (HPT) axis remains immature, maternal thyroid dysfunction can profoundly impact neurodevelopment (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we probe the potential link between maternal SCH and neurodevelopmental disorders, with a specific focus on ASD. Employing a highly standardized approach mirroring autism-specific behaviors in humans, we comprehensively assess the impact of maternal SCH during pregnancy on offspring neurodevelopment. Our findings reveal that maternal SCH during pregnancy and lactation induces ASD-like behavioral abnormalities and diminishes the number of hippocampal neurons in offspring. Furthermore, we observe decreased expression of pivotal molecules involved in hippocampal neurogenesis and synaptic plasticity, namely BDNF, CREB, and Bcl-2. Additionally, we explore the gene expression profiles of m-TOR and Wnt, given their established roles in neural development and synaptic function. In conclusion, our study, utilizing a rat model of SCH during pregnancy, sheds light on autism-like behavioral alterations in offspring, accompanied by hippocampal developmental deficits. We further elucidate the effects on the Wnt signaling pathway and the CREB-BDNF pathway, aiming to contribute to a deeper understanding of this critical area of research and provide guidance for future investigations.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eSpecific pathogen-free nulliparous female Wistar rats (n\u0026thinsp;=\u0026thinsp;24), weighing 180-200g, were used in all experimental procedures. They were housed in a climate-controlled specific-pathogen-free laboratory (temperature 25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and relative humidity 55%) with a 12-hour light/dark photoperiod. All animals were permitted free access to normal rat chow and tap water. All experimental procedures were approved by Animal Care and Use Committee at China Medical University, which complies with the National Institute of Health Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eThyroidectomy surgery and drug delivery\u003c/h2\u003e \u003cp\u003eA total of 24 female rats were randomized into control and SCH groups, with 12 rats in each group. According to our previous study (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), all surgeries were performed after intraperitoneal injection of 3% sodium pentobarbital (0.1 ml/100 g). Briefly, the anesthetized rats were fixed on the operating table, and the skin was incised along the midline of the neck, and the subcutaneous tissue and sternocleidomastoid muscle were bluntly separated. The thyroid glands were located on either side of the trachea. A midline incision was made in the isthmus, and the thyroid gland was carefully separated from the trachea with forceps while avoiding injury to the recurrent laryngeal nerve, and then penicillin was gradually injected after suturing to prevent infection. After surgery, to prevent possible hypocalcemia due to destruction of the parathyroid glands by total thyroidectomy, rats were provided with 0.1% (w/v) calcium lactate in the drinking water postoperatively and fed with normal rat chow. One month after the rats were operated, L-T\u003csub\u003e4\u003c/sub\u003e (Sigma, USA) was continuously infused as a daily subcutaneous injection of 1.0-1.05 \u0026micro;g/100g. All female rats were mated with normal male rats (female to male ratio\u0026thinsp;=\u0026thinsp;2:1). The following day, when spermatozoa were detected on microscopic vaginal smears, this day was designated as day 0 (E0) of pregnancy. Pregnant females were housed in individual cages until delivery, and the day of birth of the offspring was designated as postnatal day 0 (P0). Blood (approximately 2 ml) was drawn from all groups of female rats on days E7, E13, and P1 after injection to monitor total T\u003csub\u003e4\u003c/sub\u003e and TSH levels. The schedule of the project is shown in (Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSerum hormone levels assessments\u003c/h2\u003e \u003cp\u003eBlood samples obtained from rats were immediately centrifuged at 13,000 rpm for 15 min and stored at -80\u0026deg;C. TT\u003csub\u003e4\u003c/sub\u003e was calculated by Roche electrochemiluminescence (Roche Diagnostic Products, Los Angeles, CA, USA). TSH was determined by ELISA kit for thyroid-stimulating hormone (Cloud-Clone Corp., Houston, TX, USA). The inter- and intra-assay coefficients of variation (CVs) for TT\u003csub\u003e4\u003c/sub\u003e were 3.38\u0026ndash;4.26% and 1.37\u0026ndash;1.79%, respectively. The inter-assay and intra-assay coefficients of variation (CVs) for thyroid-stimulating hormone (TSH) were \u0026lt;\u0026thinsp;10% and \u0026lt;\u0026thinsp;12%, respectively. All samples were duplicated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral tests\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eSelf-grooming\u003c/h2\u003e \u003cp\u003eThe behavioral procedure was as described previously (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The rats were individually placed in a clean, standard open space (40 cm\u0026times;40 cm) and the experiment lasted for a total of 20 min, with the first 10 min being the acclimatization phase, during which the total time spent grooming and the total number of times the rats groomed during the second 10 min were measured. The duration of the rat's self-grooming behavior was also recorded and videotaped with a timer at a location 1.5 m away from the cage. Self-grooming was defined as licking paws, licking legs, washing nose and face, or scratching head, body, fur and tail with any paw.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eThree-chambered Social Test\u003c/h2\u003e \u003cp\u003eA total of five-week-old rats were used to determine sociality and preference for social novelty. The test consisted of three 10-min phases, including habituation, sociability, and novelty preference. Before each phase, the test mouse was maintained in the middle chamber while two doorways were closed. In the first phase, the test mouse was allowed to habituate in the three-chamber arena for 10 min. In the second sociability phase, the test mouse was exposed to an age-matched unfamiliar male mouse (stranger 1) confined in a wire cage, which was located in one of the chambers, and an identical empty wire cage was placed at the corresponding spot in the opposite chamber. In the third novelty preference phase, an age and sex-matched novel mouse (stranger 2) was enclosed in the empty wire cage, and the test mouse was allowed to roam between chambers containing familiar mouse (stranger 1) and novel mouse (stranger 2). All parameters, including the time spent in each chamber and the sniffing time with the unfamiliar rats (Stranger 1 and Stranger 2), were recorded and calculated using the automated Noldus Observer software (Ethovision 11.0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eOpen-field test (OFT)\u003c/h2\u003e \u003cp\u003eOpen-field locomotion in a novel environment was assessed as previously described(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The open-field apparatus was made of gray plywood with dimensions of 40 \u0026times; 40 cm and a wall height of 30cm. Rats were placed in the center of the open field and their movements were recorded with a video camera fixed to the top of the apparatus for 10 or 30 min and analyzed with Ethovision 11.0 (Noldus). The test apparatus was cleaned with 70% ethanol between two subjects and then wiped clean with a clean paper towel. The center area of the empty field instrument was 25% of the total area (approximately 25 cm \u0026times; 25 cm quare).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eMorris Water Maze Test (MWM)\u003c/h2\u003e \u003cp\u003eThe MWM test was performed as previously described(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Before the test, rats were allowed to swim freely in a pool without a platform for 60 seconds. For spatial learning, the rats were trained to find the underwater platform within 60 seconds on each trial. If the rats failed to find the hidden platform within 60 seconds, they were manually guided to the platform and stayed there for 30 seconds. The training was conducted 3 times a day for 4 days. On day 5 for the spatial memory test, the platform was removed and escape latency, number of platform traversals, duration in the platform quadrant, and average swimming speed were monitored using Noldus Observer software (Ethovision 11.0).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTissue Processing and Histopathology Staining\u003c/h2\u003e \u003cp\u003eThe histomorphology of the hippocampus was observed using H\u0026amp;E and Nissl staining at P44. Five to eight young rats from each group were anesthetized with 10% chloral hydrate and then transcardially perfused with cold saline and 4% paraformaldehyde (PFA) in 0.01M phosphate-buffered saline (PBS, pH 7.4) for 30 minutes. The brains were subsequently removed and stored in 4% PFA in PBS at 4\u0026deg;C overnight, followed by cryoprotection in 30% sucrose at 4\u0026deg;C. Coronal sections were embedded in paraffin, and consecutive 5-\u0026micro;m-thick coronal sections of the frontal lobes were cut and stained with H\u0026amp;E and toluidine blue (Nissl staining method). The total integrated optical density (IOD), representing the level of Nissl bodies in the CA1 region of the hippocampus, was measured at 40\u0026times; and 400\u0026times; magnification using a cast-grid microscope (MetaMorph/DP10/Bx41, UIC/OLYMPUS, US/JP) and an image analysis program (MetaMorph offline 4.65).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blotting\u003c/h2\u003e \u003cp\u003eHippocampal samples from P44 rats were collected and processed as described in our previous study. Proteomes were extracted using a BCA kit [Pierce\u0026trade;, Rockford, IL, USA], and then 0.2% bromophenol blue and 7.5% β-mercaptoethanol were added and mixed by pipetting. The proteome extracts were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in TBS-T (50 mM Tris HCl, pH 7.4, 100 mM NaCl, 0.2% Tween-20) for 30 minutes with gentle stirring. The membranes were then incubated overnight at 4\u0026deg;C with primary antibodies: BDNF (1:1,000; Cell Signaling Technology), CREB (1:1,000; Cell Signaling Technology), Bcl-2 (1:1,000; Cell Signaling Technology), and β-actin (1:1,000; Santa Cruz Biotechnology). Following incubation, the membranes were rinsed twice with distilled water, then rinsed with TBS-T, and subsequently incubated for 1 hour at room temperature with horseradish peroxidase-conjugated anti-mouse/anti-rabbit IgG (1:10,000\u0026ndash;15,000). After additional rinsing with TBS-T, the experiment was repeated five times. The optical density values of the target bands were analyzed using a gel image processing system (Bio-Rad).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was obtained from hippocampal tissue using TRIzol reagent (Thermo Fisher Scientific, USA) according to the manufacturer's instructions and its concentration was determined using a microplate reader (Molecular Devices, USA). Total RNA was then reverse transcribed to cDNA using the Revert Aid First Strand cDNA Synthesis Kit (Fermentas, USA) following the manufacturer's instructions. using the Quantitect SYBR Green PCR Kit (Thermo Fisher Scientific, USA) q-PCR was performed on an ABI PRISM 7500 Real-Time System (Applied Biosystems, USA). The amplification parameters were as follows: denaturation at 95\u0026deg;C for 30 seconds, annealing at 95\u0026deg;C for 5 seconds, extension at 60\u0026deg;C for 30 seconds, 40 cycles, and detection of signal at 60\u0026deg;C. Data were quantified using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method and normalized to β-actin expression. Primer sequences (Sangon Biotech) were: m-TOR 5'-ATCGTGCTGTTGGGTGAGAG-3' and 5'-TGGATC TCCAGCTCTCCGAA-3'; Wnt1, 5'-AACAGTAGTGGCCGATGGTG-3' and 5'- GGGTTCTGTCGGGATCAGTCG-3'; β-actin, 5'-GGCTGTATTCCCCTCCATCG-3' and 5'-CCAGTTGGTAACAATG CCATGT-3'.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM). All statistical analyses were performed using SPSS software. Comparisons were analyzed using one-way ANOVA followed by Tukey's test. One-way repeated ANOVA was used for the escape latency data of MWM. All graphs were analyzed using Graph Pad Prism 8 (GraphPad Software Inc.). Statistical significance was defined at *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eThyroid Hormone Levels in Maternal Rats\u003c/h2\u003e \u003cp\u003eTo assess the success of maternal SCH model, we measured serum TT4 and TSH levels on days 7 and 13 of pregnancy and on the day of delivery. The results showed that TSH levels were significantly higher in the SCH group compared to the control group (on all days tested; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but TT\u003csub\u003e4\u003c/sub\u003e levels were not statistically different from the control group. The results met the criteria for SCH and confirmed the successful establishment of the maternal SCH rat model. The mean body weights of the littermates at P40 were as follows: control group, 122.11\u0026thinsp;\u0026plusmn;\u0026thinsp;3.66 g; SCH group, 117\u0026thinsp;\u0026plusmn;\u0026thinsp;4.75 g. There was no significant difference(Table\u0026nbsp;1, Fig .2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSCH offspring rats show increased stereotyped behaviors\u003c/h2\u003e \u003cp\u003eWe evaluated the stereotypic behaviors of offspring rats by recording the frequency and duration of behaviors like licking paws, washing nose and face, or scratching fur with feet during their free-range activities (Fig.\u0026nbsp;3A). The results showed that the frequency and duration of self-grooming stereotypic behaviors increased significantly in male rats in the SCH group compared to control rats (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the total distance traveled during these activities did not change significantly (Fig.\u0026nbsp;3B). In the female group, there was a statistically significant increase in the frequency of stereotypic behaviors in the SCH group compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), although no significant difference was observed in the duration and total distance traveled during the activity (Fig.\u0026nbsp;3C). These findings indicate that SCH offspring rats exhibited more frequent grooming behaviors, which are suggestive of typical autistic behaviors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSocial interaction deficits in SCH offspring rats\u003c/h2\u003e \u003cp\u003eThe social activity behaviors of the two groups of offspring rats were examined using a three-chambered social test (Fig.\u0026nbsp;4A, B). Our study found that male rats in the control group spent significantly more time sniffing Stranger Rat 1 than the empty cage (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that the control group males did not have significant social behavioral deficits. However, male rats in the SCH group showed no significant difference in sniffing time between Stranger Rat 1 and the empty cage, suggesting that they exhibited significant social behavioral deficits. Similarly, in the social novelty preference and motivation tests, control group males spent significantly more time sniffing Stranger Rat 2 than Stranger Rat 1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), demonstrating normal social novelty preference behavior. In contrast, male rats in the SCH group did not show a significant difference in sniffing time between Stranger Rat 1 and Stranger Rat 2, indicating ASD-like behaviors such as novelty preference deficits and decreased socialization (Fig.\u0026nbsp;4C).\u003c/p\u003e \u003cp\u003eIn the female group, there was no significant difference between the SCH group and the control group in the time spent sniffing Stranger 1 and the empty cage. In the social novelty preference test, only the control group females showed normal social novelty preference behavior, spending significantly more time sniffing Stranger Rat 2 than Stranger Rat 1(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, female rats in the SCH group showed no significant difference in sniffing time between Stranger Rat 1 and Stranger Rat 2, indicating a significant social novelty preference deficit (Fig.\u0026nbsp;4D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eOpen-field test (OFT)\u003c/h2\u003e \u003cp\u003eIn order to exclude the possibility that locomotor dysfunction may lead to social deficits, we assessed motor performance in the open field test (Fig.\u0026nbsp;5A), in which a statistically significant difference was observed in the SCH male group compared to the control group in terms of the time spent in the central area of the open field (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting the presence of anxious behaviours in the SCH male offspring rats (Fig.\u0026nbsp;5B). In both female groups, no significant difference was observed in the time spent in the central area of the open field in the SCH group compared to the control group. In addition, there was no significant difference in the total distance travelled between the male and female groups. (Fig.\u0026nbsp;5C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eSpatial learning and memory impairment in SCH offspring rats\u003c/h2\u003e \u003cp\u003eAutism is often accompanied by intellectual disabilities and memory impairment, so we conducted Morris Water Maze (MWM) tests to assess these aspects. The rats were divided into cages based on sex, and the experiment was conducted exclusively on male offspring rats (Fig.\u0026nbsp;6A). On the first day of the spatial learning experiment, the rats were in the acclimatization period, so there was no significant difference in the mean escape latency (i.e., the time taken to reach the hidden platform) between the groups. From the second day of training, the mean escape latency of the rats in each group decreased as the number of training sessions increased. However, from the second to the fifth day, the SCH group showed a slower decrease in escape latency and spent significantly more time searching for the hidden platform compared to the control group. ANOVA indicated that this difference was statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;6B). On the last day, during the probe trials phase, the SCH group spent significantly less time in the target quadrant compared to the control group, with the difference being statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;6C). Additionally, the frequency of crossing the platform area was significantly lower in the SCH group, and this difference was also statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;6D). However, there was no significant difference in the average swimming speed between the SCH group and the control group, suggesting that the SCH group exhibited impaired spatial learning and memory (Fig.\u0026nbsp;6E).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDamage of SCH to neurons in the hippocampal CA1 region of offspring rats\u003c/h2\u003e \u003cp\u003eRed arrows point to representative Nissl bodies. The IOD values of Nissl bodies in the hippocampal CA1 region of SCH male offspring rats were significantly reduced compared to those of the control group. The Nissl bodies in the SCH group were either broken and missing, or the neurons were irregularly arranged and poorly delaminated. These results suggest that maternal SCH causes dissolution and loss of Nissl bodies in hippocampal neurons of male offspring, disrupting neuronal structure and function (Fig.\u0026nbsp;7A, B). Compared to the normal control, statistically significant differences were observed in the IOD of Nissl bodies in the CA1 region of the hippocampus in the male group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01, Fig.\u0026nbsp;7C), while no significant difference was seen in the IOD of the female group (Fig.\u0026nbsp;7D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eEffect of SCH on hippocampal CREB, BDNF, and Bcl-2 levels in offspring rats\u003c/h2\u003e \u003cp\u003eWe observed that hippocampal CREB levels were significantly decreased in male offspring rats in the SCH group compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, we investigated whether BDNF, a downstream molecule of CREB, mediated the effects of maternal SCH on autism-like behaviors in offspring rats. The results revealed that hippocampal BDNF levels were significantly lower in male offspring rats of the SCH group compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, Bcl-2 expression was also significantly decreased in the SCH male group, with the difference being statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, hippocampal CREB and BDNF levels were significantly decreased in female offspring rats of the SCH group compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, while Bcl-2 expression was slightly decreased in the female SCH group, the difference was not statistically significant (Fig.\u0026nbsp;8A, B).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eEffect of SCH on m-TOR and Wnt gene expression in offspring rats\u003c/h2\u003e \u003cp\u003eIn male offspring rats of the SCH group, the expression of the target gene m-TOR was significantly elevated compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Conversely, Wnt expression was significantly lower in the SCH male group compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In female offspring rats, m-TOR expression was slightly elevated in the SCH group compared to the control group, but this difference was not statistically significant. Similarly, Wnt gene expression was slightly decreased in the SCH female group compared to the control group, but again, the difference was not statistically significant (Fig.\u0026nbsp;9A, B).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study is the first to directly investigate the molecular mechanisms by which maternal SCH induces autism-like behaviors in offspring. The key findings of this study are as follows: First, SCH during gestation and lactation induces autism-like behaviors in male offspring rats, such as increased grooming behavior and social interaction deficits. Second, the integrated optical density of Nissl staining in the CA1 region of the hippocampus was lower in male offspring of SCH dams compared to controls. Third, maternal SCH led to a significant reduction in hippocampal BDNF, CREB, and Bcl-2 protein levels, a significant up-regulation of m-TOR gene expression, and a significant down-regulation of Wnt gene expression in the offspring compared to sham-operated control groups. Additionally, we observed sex-specific phenotypic differences. In conclusion, this study provides direct evidence that maternal SCH results in motor and/or social interaction impairments in offspring. The Wnt signaling pathway, along with thyroid-regulated genes BDNF, CREB, and Bcl-2, play critical roles in the pathogenesis of autism-like behavioral abnormalities in the offspring of SCH mothers.\u003c/p\u003e \u003cp\u003eOver the past decade, increasing evidence has shown that maternal subclinical hypothyroidism (SCH) impairs learning and memory functions in offspring(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The hippocampus is a vital brain region in the central nervous system (CNS) involved in regulating emotions and memory(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). It contains a high density of thyroid hormone receptors and various neurotransmitters(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) and serves as a significant regulator of the HPT axis(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The cornu ammonis (CA) region of the hippocampus, primarily composed of pyramidal cells, is crucial for processing emotional information(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) and forming short-term memory(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). cAMP response element-binding protein (CREB) is a nuclear transcription factor that plays a pivotal role in neuronal development, including neuronal survival, proliferation, synapse formation, synaptic plasticity, and long-term memory formation(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). CREB-dependent transcription is critical for various forms of learning and memory(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Previous research has shown that iodine deficiency leads to a reduction in total CREB and phosphorylated CREB (p-CREB) levels in the hippocampus of lactating and adolescent rats(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). In this study, we found a decrease in CREB protein levels in the offspring of maternal SCH.\u003c/p\u003e \u003cp\u003eBrain-derived neurotrophic factor (BDNF), a downstream target of CREB(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), is synthesized by neurons and astrocytes and plays a crucial role in the development and survival of neural stem cells (NSCs) by binding to TrkB receptors(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Our findings demonstrated a significant reduction in hippocampal BDNF protein levels in the offspring of maternal SCH, consistent with previous studies(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Alterations in BDNF are linked to core psychopathological features of autism, as BDNF is essential for synaptic plasticity and cognitive functions(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Since CREB acts as a transcriptional activator of BDNF through motifs preceding BDNF exon IV(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), the down-regulation of BDNF proteins in the hippocampus of the offspring may be mediated by reduced CREB activity(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Impaired CREB-BDNF signaling has been widely implicated in the pathogenesis of autism(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), and our results further support the disruption of this signaling cascade in the context of maternal SCH.\u003c/p\u003e \u003cp\u003eThyroid hormone can directly activate the apoptotic pathways involving the Bcl-2 family, in addition to influencing the brain neurotrophic factor pathway(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Bcl-2 is crucial for neuronal survival and apoptosis regulation(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Research by Kim et al. and Jang et al. has demonstrated that thyroid hormone deficiency induces neuronal apoptosis in rat models(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Dysregulation of anti-apoptotic Bcl-2 family members has been implicated in mood disorders such as anxiety, depression, and bipolar disorder in humans. Moreover, deletion of the Bcl-2 gene in mouse neural progenitor cells has been associated with anxiety-like behavior in rats(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Our study reveals that maternal SCH significantly reduces Bcl-2 protein expression in the brains of offspring rats. Although we did not perform apoptosis staining, it is plausible that apoptosis could occur and be completed during gestation(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The complexity of apoptotic processes necessitates further in-depth studies to fully elucidate the role of Bcl-2 dysregulation in the context of maternal SCH and its effects on offspring brain development.\u003c/p\u003e \u003cp\u003emTOR is a crucial effector protein downstream of the PI3K/Akt pathway(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), integrating multiple extracellular signals involved in protein synthesis and synaptic plasticity(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Brain organoid culture experiments by Takei et al. demonstrated that abnormalities in the PI3K/AKT/mTOR signaling pathway affect cortical neurogenesis and induce autism-like behavioral changes(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). mTOR promotes synaptic plasticity and memory by regulating protein synthesis(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), and mutations in its pathway components are widely associated with neurodevelopmental disorders(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). For instance, peripheral blood mononuclear cells from patients with mild and severe idiopathic autism exhibit heightened mTOR signaling activity, suggesting it as a molecular signature of clinical ASD severity(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Consistent with previous reports, our study showed that male offspring of the SCH group displayed autism-like behavioral phenotypes, with a significant upregulation of mTOR gene expression in the hippocampus. Notably, the mTOR-dependent protein translation process in the cytoplasmic lysate of neural circuits is regulated by axonal BDNF/TrkB signaling, providing a plausible explanation for our findings(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExisting literature underscores the critical role of the Wnt pathway in neurodevelopmental disorders(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Disruption of Wnt signaling during the embryonic development of the cerebral cortex has been linked to delayed neuronal migration and abnormal gyrus connectivity(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), leading to a range of neurodevelopmental disorders such as ASD(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). In the present study, we observed significant downregulation of the Wnt gene in the male offspring of SCH rats, along with behavioral abnormalities such as sensory dysfunction, social deficits, and obsessive-compulsive behaviors. These findings suggest that dysregulation of the Wnt pathway may be one mechanism by which SCH leads to the development of neuropsychiatric disorders in offspring. Additionally, Liu et al. have associated the PI3K-Akt-Wnt pathway with improvements in short-term memory following exercise(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), while Park et al. have elucidated the relationship between BDNF expression and Wnt signaling(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Wang et al. and Yang et al. have demonstrated the importance of Wnt signaling in neural development and its regulatory relationship with BDNF(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). These studies emphasize the importance of the Wnt pathway and BDNF signaling in neurodevelopmental disorders, especially considering the autism-like behaviors observed in male rats, consistent with our findings.\u003c/p\u003e \u003cp\u003eIt is well-documented that the prevalence of ASD is significantly higher in males than in females(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), indicating that innate biological factors influence the manifestation of neurodevelopmental disorders differently between the sexes(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Both genetic and hormonal factors contribute to these gender differences. Several studies have identified sex-specific genetic variants associated with ASD, with specific genes on the X and Y chromosomes potentially contributing to the higher male prevalence(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e).\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIn conclusion, our study provides evidence that maternal subclinical hypothyroidism (SCH) influences the Wnt pathway and brain-derived neurotrophic factor (BDNF) levels in offspring rats, with particularly pronounced effects in males. These sex-specific findings, including autistic-like behaviors, underscore the potential neurodevelopmental impact of maternal SCH.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS AND DISCLOSURES\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by The National Natural Science Foundation of China (Grant: 81200573 and 82103338). Liaoning Province Science and Technology\u0026nbsp;Planning\u0026nbsp;Project (Grant 2021JH2/10300108)\u003c/p\u003e\n\u003cp\u003eThe authors report no biomedical financial interests or potential conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eShan ZY, Chen YY, Teng WP, Yu XH, Li CY, Zhou WW, \u003cem\u003eet al.\u003c/em\u003e (2009): A study for maternal thyroid hormone deficiency during the first half of pregnancy in China. \u003cem\u003eEur J Clin Investigation\u003c/em\u003e 39: 37\u0026ndash;42.\u003c/li\u003e\n \u003cli\u003eSealey LA, Hughes BW, Sriskanda AN, Guest JR, Gibson AD, Johnson-Williams L, \u003cem\u003eet al.\u003c/em\u003e (2016): Environmental factors in the development of autism spectrum disorders. \u003cem\u003eEnvironment International\u003c/em\u003e 88: 288\u0026ndash;298.\u003c/li\u003e\n \u003cli\u003eBaron-Cohen S, Auyeung B, N\u0026oslash;rgaard-Pedersen B, Hougaard DM, Abdallah MW, Melgaard L, 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288.\u003c/li\u003e\n \u003cli\u003eChoi GB, Yim YS, Wong H, Kim S, Kim H, Kim SV, \u003cem\u003eet al.\u003c/em\u003e (2016): The maternal interleukin-17a pathway in mice promotes autism-like phenotypes in offspring. \u003cem\u003eScience\u003c/em\u003e 351: 933\u0026ndash;939.\u003c/li\u003e\n \u003cli\u003eBohmwald K, Andrade CA, Mora VP, Mu\u0026ntilde;oz JT, Ram\u0026iacute;rez R, Rojas MF, Kalergis AM (2022): Neurotrophin Signaling Impairment by Viral Infections in the Central Nervous System. \u003cem\u003eIJMS\u003c/em\u003e 23: 5817.\u003c/li\u003e\n \u003cli\u003eWerling DM, Geschwind DH (2013): Sex differences in autism spectrum disorders: \u003cem\u003eCurrent Opinion in Neurology\u003c/em\u003e 26: 146\u0026ndash;153.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Subclinical hypothyroidism hippocampus ASD offspring Rat","lastPublishedDoi":"10.21203/rs.3.rs-4675145/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4675145/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEpidemiological studies have illuminated that abnormal maternal thyroid function in early pregnancy escalates the risk of autism spectrum disorder (ASD) in offspring by nearly fourfold. However, the exact mechanism by which maternal thyroid dysfunction affects the risk of ASD in the offspring remains unknown. Early pregnancy emerges as a pivotal juncture for fetal brain development, with the fetus heavily reliant on maternal thyroid hormones for its neurological maturation. Our investigation unveils a striking correlation: mothers afflicted by perinatal subclinical hypothyroidism (SCH) tend to have male offspring displaying marked ASD-like behavioral anomalies, typified by conspicuous diminution in social interactions and repetitive behavioral patterns. Furthermore, we discerned a substantial reduction in neuron count within critical brain regions among offspring of SCH mothers, suggestive of underlying neurodegeneration or developmental impediments. Moreover, diminished levels of brain-derived neurotrophic factor (BDNF), cAMP response element binding protein (CREB), and Bcl-2 were noted in the hippocampal region of SCH offspring, juxtaposed with a noteworthy upregulation of mTOR expression and downregulation of Wnt. These findings coalesce to suggest that the Wnt-mediated signaling pathway, in concert with its interplay with the downstream target BDNF, might underpin one of the mechanisms by which maternal SCH predisposes offspring to autism-like behaviors.\u003c/p\u003e","manuscriptTitle":"Wnt/BDNF pathway mediates autism-like behaviors induced by prenatal subclinical hypothyroidism in male neonatal rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 12:48:44","doi":"10.21203/rs.3.rs-4675145/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-12-04T10:22:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-10-19T14:21:32+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-10-05T16:23:22+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-09-28T01:18:33+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-09-25T05:08:13+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-09-19T07:26:00+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-09-19T01:54:32+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-09-18T21:23:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-03T10:23:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-02T14:54:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Psychiatry","date":"2024-07-02T14:54:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"238956d3-14a4-4029-9179-0735d32a310f","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34065863,"name":"Health sciences/Diseases"},{"id":34065864,"name":"Biological sciences/Genetics"}],"tags":[],"updatedAt":"2025-10-08T07:05:32+00:00","versionOfRecord":{"articleIdentity":"rs-4675145","link":"https://doi.org/10.1038/s41398-025-03570-6","journal":{"identity":"translational-psychiatry","isVorOnly":false,"title":"Translational Psychiatry"},"publishedOn":"2025-10-07 04:00:00","publishedOnDateReadable":"October 7th, 2025"},"versionCreatedAt":"2024-08-09 12:48:44","video":"","vorDoi":"10.1038/s41398-025-03570-6","vorDoiUrl":"https://doi.org/10.1038/s41398-025-03570-6","workflowStages":[]},"version":"v1","identity":"rs-4675145","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4675145","identity":"rs-4675145","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}