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

doi:10.21203/rs.3.rs-4675145/v1

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

https://doi.org/10.21203/rs.3.rs-4675145/v1

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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

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.

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-T₄ (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 T₄ 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. TT₄ 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 TT₄ 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.

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 TT₄ 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).

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).

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.

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.

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Table 1 is available in the Supplementary Files section

The authors have declared there is NO conflict of interest to disclose

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

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Animals

Thyroidectomy surgery and drug delivery

Serum hormone levels assessments

Behavioral tests

Self-grooming

Three-chambered Social Test

Open-field test (OFT)

Morris Water Maze Test (MWM)

Tissue Processing and Histopathology Staining

Western Blotting

Quantitative real-time PCR

Statistical analysis

RESULTS

Thyroid Hormone Levels in Maternal Rats

SCH offspring rats show increased stereotyped behaviors

Social interaction deficits in SCH offspring rats

Open-field test (OFT)

Spatial learning and memory impairment in SCH offspring rats

Damage of SCH to neurons in the hippocampal CA1 region of offspring rats

Effect of SCH on hippocampal CREB, BDNF, and Bcl-2 levels in offspring rats

Effect of SCH on m-TOR and Wnt gene expression in offspring rats

DISCUSSION

CONCLUSIONS

Declarations

References

Table

Additional Declarations

Supplementary Files

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