Behavioral abnormalities and phosphorylation deficits of extracellular signal-regulated kinases 1 and 2 in rat offspring of the maternal immune activation model

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Behavioral abnormalities and phosphorylation deficits of extracellular signal-regulated kinases 1 and 2 in rat offspring of the maternal immune activation model Wenqiang Li , Fuping Sun , Xiaoge Guo , Yunqing Hu , Shuang Ding , Minli Ding , Meng Song , Minglong Shao , Yongfeng Yang , Weiyun Guo , Luwen Zhang , Yan Zhang , Xiujuan Wang , Xi Su , Luxian Lv PII: DOI: Reference:

S0031-9384(19)30680-8 https://doi.org/10.1016/j.physbeh.2020.112805 PHB 112805

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Physiology & Behavior

Received date: Revised date: Accepted date:

3 July 2019 23 December 2019 14 January 2020

Please cite this article as: Wenqiang Li , Fuping Sun , Xiaoge Guo , Yunqing Hu , Shuang Ding , Minli Ding , Meng Song , Minglong Shao , Yongfeng Yang , Weiyun Guo , Luwen Zhang , Yan Zhang , Xiujuan Wang , Xi Su , Luxian Lv , Behavioral abnormalities and phosphorylation deficits of extracellular signal-regulated kinases 1 and 2 in rat offspring of the maternal immune activation model, Physiology & Behavior (2020), doi: https://doi.org/10.1016/j.physbeh.2020.112805

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Highlights 

MIA induces progressive worsening behaviors in adolescents and adults offspring.

 

MIA impaires ERK activation in offspring. MIA induces age- and region-related alterations of neurofilaments in offspring.

Behavioral abnormalities and phosphorylation deficits of extracellular signal-regulated kinases 1 and 2 in rat offspring of the maternal immune activation model Wenqiang Lia,b,c,1, Fuping Suna,b,c,1, Xiaoge Guoa,b,c, Yunqing Hua,b,c, Shuang Dinga, Minli Dinga, Meng Songa,b,c, Minglong Shaoa,b,c, Yongfeng Yanga,b,c, Weiyun Guod, Luwen Zhanga,b,c, Yan Zhanga, Xiujuan Wanga, Xi Sua,b,c*, Luxian Lva,b,c*. a Henan Mental Hospital, The Second Affiliated Hospital of Xinxiang Medical University, Xinxiang, China. b Henan Key Lab of Biological Psychiatry of Xinxiang Medical University, Xinxiang, China. c International Joint Research Laboratory for Psychiatry and Neuroscience of Henan, Xinxiang, China. d College of Life Science and Technology, Xinxiang Medical University 1

Wenqiang Li and Fuping Sun contributed equally to this work. Corresponding author: Xi Su, Luxian Lv. Department of Psychiatry of the Second Affiliated Hospital of Xinxiang Medical University, No.388, Jianshe Middle Road, Xinxiang, 453002, China. Tel:3373969. E-mail: [email protected]; [email protected]. *

Highlights MIA induces progressive worsening behaviors in adolescents and adults offspring. MIA impaires ERK activation in offspring. MIA induces age- and region-related alterations of neurofilaments in offspring. Abstract Extracellular signal-regulated kinase (ERK) signal transduction is known to be associated with neurogenesis and neuronal differentiation and as such may be related to the synaptic plasticity associated with cognitive function. Although antipsychotic drug studies have suggested a potential role for the ERK cascade in schizophrenia, the mechanistic basis is unknown. The maternal immune activation (MIA) rat model is a well-known to simulate many of the clinical symptoms of schizophrenia, including cognitive deficits, but a role in this model for dynamic changes in ERK has not been established. In this study, polyinosinic:polycytidylic acid was administered to rats intravenously at a dose of 10 mg/kg on embryonic day 9.5 to produce MIA. The effect of MIA on behavior and ERK phosphorylation within the prefrontal cortex and the hippocampus of adolescent and adult offspring were explored. We also examined neurofilaments, a marker of neurogenesis, which have been reported to be modulated by ERK signaling. The results demonstrate an age- and

region-specific profile of ERK expression and phosphorylation and suggest possible relationships among ERK, neurofilament expression, and cognitive performance in schizophrenia. Keywords: Extracellular signal-regulated kinases; Cognitive impairment; schizophrenia; Synaptic plasticity. 1. Introduction Schizophrenia is a common and severe neurodevelopmental disorder that affects almost 1% of the population. The disorder manifests as a disruption in cognition and emotion with negative and positive symptoms. Cognitive impairment is a key symptom of most schizophrenic patients1 and is related to functional outcomes and long-term prognosis 2-5. Cognitive deficit treatment is mainly comprised of second generation (atypical) drugs (SGAs)6-9. The mechanistic basis for the effect of SGAs in patients with cognitive deficits is unknown. Animal models offer a means by which to evaluate the pathogenesis of schizophrenia and as well the basis for pharmacologic intervention. The maternal immune activation (MIA) model of schizophrenia was developed based on epidemiological evidence. MIA adults offspring that have altered brain structure, morphology, electrophysiology, and neurochemistry, as well as changes in behavior similar to the pathological features of schizophrenia10,11. Experimentally, one commonly used approach to induce MIA is to administer pregnant animals the double-stranded RNA analogue polyinosinic:polycytidylic acid (poly I:C), which produces an effect similar to that induced by viral infection10. Numerous studies have shown that the offspring of pregnant dams treated with poly I:C show a battery of schizophrenia-like behaviors including cognitive deficits12,13 as well as neuro-immunological14 and brain morphological abnormalities 15. The poly I:C MIA model is a powerful tool for the investigation of schizophrenia. Mechanistic evaluation of SGAs has provided for the development of novel therapeutic strategies for the treatment of schizophrenia. Through modulation of neurotransmitter systems,

an increasing number of mechanistic pharmacological studies have shown SGAs to impact various features of schizophrenia, offering potential new targets for treatment 16. Undoubtedly, the effects of SGAs will depend in part on regulation of various signaling pathways. For example, extracellular signal-regulated kinases 1 and 2 (ERK1/2) are among the attractive targets by which SGAs may impact schizophrenia16,17. Pharmacological studies have demonstrated clozapine to regulate ERK1/2 signaling in the mouse prefrontal cortex (PFC) and striatum16,18. Long-term exposure to olanzapine, an analogue of clozapine, can upregulate ERK1/2 phosphorylation in the rat PFC19. The ERK1/2 pathway is related to cognitive and learning functions by participation in synaptic plasticity and activation of proteins associated with dendritic organization, neuronal survival and differentiation, synaptogenesis, and neurotransmitter release20,21. Despite its role in synaptic plasticity, the involvement of the ERK pathway in schizophrenia has not been thoroughly examined, nor has its relationship to cognitive deficits in schizophrenia been clarified. Neurofilaments are a major component of the cytoskeleton, providing support for axons as well as regulation of their diameter, which affects neuron morphology, plasticity, and signal transduction22-24. In mammals, neurofilaments are composed of three proteins; neurofilament protein L (low molecular weight; NFL), M (medium molecular weight, NFM), and H (high molecular weight, NFH). The ERK pathway has been associated with neurofilaments as well as neurite retraction in vivo25-29, with validation of these associations in animal disease models essential. Although pharmacological studies have demonstrated involvement of ERK1/2 in antipsychotic action and in cognitive rescue, dynamic changes in ERK1/2 remain poorly understood in models of schizophrenia. Likewise the importance of neurofilaments in schizophrenia requires further investigation in that neurofilaments are ERK1/2 targets and are related to synaptic plasticity25,26. Herein, the MIA schizophrenia rat model induced by poly I:C exposure at embryonic day 9.5 (E9.5) was used to study the dynamic changes in ERK and neurofilaments in the hippocampus (HIP) and prefrontal cortex (PFC) (two brain regions

related to cognitive performance) in adolescent and adult rat offspring at postnatal day 40 (PND40) and PND60. 2. Materials and methods 2.1. Animals Male and female Sprague-Dawley rats for mating were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animals were housed at 22 ± 2°C in 57% ± 2% relative humidity, under a 12/12-h light/dark cycle. Food and water were provided ad libitum. All animal handling and experiments were approved by the Laboratory Animal Care and Use Committee of the Henan Key Laboratory of Biological Psychiatry (Xinxiang, China). 2.2. MIA induction Male and female rats were mated overnight. The presence of a vaginal plug the next morning was recorded as E0.5. Pregnant rats received injections of either saline or 10 mg/kg poly I:C (Sigma-Aldrich, St. Louis, MO, USA) intravenously on E9.5. Neonates were allowed to be born naturally. Offspring of saline injected rats were referred to as control offspring and offspring of poly I:C-injected dams were designated MIA offspring. A total of 12 pregnant rats were evenly and randomly selected for poly I:C and vehicle administration. 2.3. Allocation and testing of offspring Pups were weaned on PND21. Control offspring and MIA offspring of the same sex were separated and maintained at 3–5 rats per cage. We only selected male pups for the experiments. Offspring from each group were divided into two groups and were undisturbed until PND40 or PND60, which corresponded respectively to adolescence and young adulthood in humans. Corresponding male offspring at each time point underwent behavioral testing and were then anesthetized with isoflurane and euthanatized for western blot analysis of ERK1/2 and neurofilaments.

2.4. Behavioral tests Y maze and Pre-pulse inhibition (PPI) tests were carried out to assess the cognitive status of MIA offspring. All behavioral procedures were carried out in the dark phase of the light-dark cycle. After each trial, the apparatus was cleaned with 75% alcohol and dried before the next test began. 2.4.1. Y maze test A spatial novelty preference task in the Y-maze was designed to evaluate spatial recognition memory. The experimental apparatus consisted of three identical black iron plate arms spaced 120° apart from each other and radiating from a central triangle, which were designated start arm, familiar arm (with a red label), and novel arm (with a yellow label). The test in the Ymaze consisted of a sample phase and a choice phase. The rats were first allowed to explore the start arm and familiar arm for 10 min, while access to the novel arm was blocked by a partition. The barrier was removed 2 h after the start of the sample phase. At that time, the animals were placed at the end of the start arm facing the central triangle and allowed to freely explore for 5 min. Entries into the novel arm and time spent there were video-recorded and analyzed using Spain Panlab Smart 3.0. Spatial recognition memory was analyzed by comparing the animal’s exploration of all three arms. 2.4.2. PPI test The PPI test was conducted using four sound-attenuated chambers. All test sessions were performed in a single-chamber startle apparatus (QMC-I, Kunming Institute of Zoology, Chinese Academy of Sciences, China). After adapting for 5 min with a background noise of 70 dB, the rats received five startle trials (120-dB burst of white noise lasting 20 ms). The rats then received three types of PPI trials (differing in pre-pulse intensity) as well as no-stimulus trials (ten times each). The PPI trials were composed of a pre-pulse stimulus of white noise at 75, 80, or 85 dB for 20 ms, followed by a 100 ms delay, and then a 40-ms stimulation of the startle reflex with white noise at 120 dB. A total of 40 stimulus presentations were randomly

distributed. The session closed with another five startle stimuli. All trials were separated by an inter-trial interval of 15 s on average (7–23 s in duration). The results of pre-pulse stimulation at 75 dB (PP75), PP80, and PP85 were calculated automatically by the system software. The PPI percentage induced by each pre-pulse intensity was calculated as [1-(startle amplitude on pre-pulse trial)/(startle amplitude on pulse alone)] × 100%. 2.5. Immunoblotting analysis Rats were sacrificed for tissue collection 2 days after behavioral testing. Briefly, rats were anesthetized with isoflurane and then underwent rapid decapitation. The HIP and PFC were separately extracted and homogenized in radioimmunoprecipitation assay buffer containing phenylmethanesulfonyl fluoride (1:100, ST506-2, Beyotime Biotechnology) and phosphatase inhibitors (1:100, P1260, Solarbio), then centrifuged at 12,000 × g for 10 min at 4°C. Supernatants were collected and the bicinchoninic acid method was used to assay protein concentration. The same amount of protein (40 μg per lane for all samples) was resolved on SDS-12% polyacrylamide gels, and proteins were electrophoretically transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Following transfer onto membranes, blots of ERK1/2 and neurofilaments were blocked with 5% non-fat milk for 2 h, while blots of p-ERK1/2 were blocked with 1% BSA for 2 h. Membranes were incubated overnight at 4°C with the appropriate primary antibody. After washing three times at 10 min/wash, the membranes were incubated with the appropriate secondary antibody for 1 h and then washed three times at 10 min/wash before visualization using an Enhanced Chemiluminescence Plus detection kit (GE RPN2232). 2.6. Antibodies Western blot analysis was performed with the following primary antibodies raised against target proteins: anti-ERK1/2 (1:2,000, AF0155, Affinity), anti-p-ERK1/2 (1:1,000, AF1014, Affinity), anti-NFM (1:1,000, ab7794, Abcam), anti-NFH (1:10,000, ab8135, Abcam), and anti-β-actin (1:200, BM0627, Boster). The following secondary horseradish peroxidase

(HRP)-conjugated antibodies were used at 1:5000 dilution: goat anti-rabbit HRP (12–348) (Millipore, Watford, UK) and goat anti-mouse (sc-2005) (Santa Cruz, Insight Biotechnology, Wembley, UK). 2.7. Statistical analysis Data are presented as means ± standard error of the mean (SEM) and were analyzed with SPSS 20.0 (IBM, Armonk, NY, USA). The relative mean value of the control group in the immunoblotting analyses was set at 100, and data from MIA offspring were expressed as percentages of the mean values obtained from offspring of saline-treated dams. The phosphorylation of ERK1/2 was expressed as a ratio of phosphorylated ERKs to total ERKs (pERKs/tERKs). The protein expression (western blot) data were analyzed using independent Student’s t-tests (two-tailed). Two-way analysis of variance (ANOVA) was used for behavioral data analysis (Y-maze). Two-way repeated measures ANOVA was used for behavioral data analysis (PPI test), followed by the Bonferroni correction for the chosen group comparisons. A probability level less than 0.05 was accepted as significant. 3. Results 3.1. Cognitive performance of MIA offspring Poly I:C (10 mg/kg) or saline was administered on E9.5 to female rats and the functional consequences of immunostimulant treatment evaluated in MIA offspring using a battery of behavioral tests at PND40 and PND60, which represent adolescence and adulthood, respectively. Y maze and PPI tests were carried out to assess cognitive status. Overall, MIA led to no significant deficit in cognitive tasks among the offspring on PND40. In adulthood, on PND60, MIA offspring showed a decrease in the number of entries into the novel arm (Fig. 1A) and time spent in the novel arm (Fig. 1B) for the Y maze test. For the PPI test, MIA offspring displayed defects at 75, 80, and 85 dB (Fig. 1C and 1D). Taken together, these results demonstrate early gestational immune activation to result in cognitive deficits in adult animals but not in adolescent pups.

3.2. Effect of MIA on ERK1/2 in the PFC and the HIP of MIA offspring The ERK1/2 cascade plays an important role in integrating multiple signaling pathways that affect neuronal structural plasticity and thereby alter cognitive performance. Therefore, alteration of ERK1/2 in the PFC and HIP of MIA offspring was assessed. The phosphorylation ratio of ERK1/2 in the PFC (Fig. 2) and the HIP (Fig. 3) at PND40 and PND60 were analyzed. During adolescence, decreases in the phosphorylation ratio of ERK1 and ERK2 was observed in the HIP (Fig. 3), with no significant change in the PFC (Fig. 2). During adulthood, ERK1 and ERK2 phosphorylation declined significantly in both the PFC and the HIP (Fig. 2 and 3). 3.3. Effect of MIA on neurofilaments in the PFC and the HIP of MIA offspring The ERK pathway has been associated with neurofilaments25,26, which are involved in structural plasticity and signal transduction. Dynamic changes in neurofilaments were evaluated in the rat MIA model as protein expression of NFM and NFH in the PFC and the HIP on PND40 and PND60. In the PFC, MIA offspring had a significant increase in NFH with only a numerical increase in NFM at PND40 (Fig. 4). There was no difference between NFM and NFH in the PFC at PND60. In the HIP, NFM protein expression was significantly increased at PND40 (Fig. 5) with only a numerical increase at PND60. NFH expression was non-significantly increased at either time points and appeared to decline with increasing age. 3.4. Immunofluorescence studies Consistent with western blot results, offspring of rats exposed to Poly I:C on GD9.5 had reduced p-ERK immunoreactivity within the PFC (Fig. 6A) and the HIP (Fig. 6B) compared to control groups at PND40 and PND60. In the PFC (Fig. 6C), no significant difference was found in NFM staining compared to control groups at PND40 and PND60. The level of NFH immunoreactivity for MIA offspring was markedly higher than control groups at PND40 with no obvious difference at PND60.

The results of NFM and NFH staining of the PFC are matched well with the results of western blots. In the HIP (Fig. 6D), pups exposed to poly I:C exhibited increased NFM immunoreactivity on PND40 compared to controls. Though no significant difference was found in total NFM fluorescence intensity compared to control at PND60, the distribution of NFM in the HIP showed a decrease in the CA3 region and an increase in the DG region. No significant difference for NFH in the HIP was observed compared to controls at PND40 or PND60.

Fig. 1. Cognitive performance in offspring of MIA rats. Cognitive behavior was analyzed in adolescent and adult offspring of rat dams intravenously injected with poly I:C (10 mg/kg) or saline on E9.5. (A) Y-maze entries into the novel arm. (B) Time spent in the Y-maze novel arm. (C) PPI performance on PND40 at 75, 80, and 85 dB. (D) PPI performance on PND60 at 75, 80, and 85 dB; n = 15 for each group. Data are means ± SEM. *P＜0.05, **P＜0.01, ＜0.001.

***

P

Fig. 2. Effect of MIA on ERK1/2 phosphorylation in the PFC of rat offspring. (A) Representative images of ERK1/2 phosphorylation in adolescent and adult offspring of rat dams intravenously injected with poly I:C (10 mg/kg) or saline on E9.5. (B) Statistical analysis of the ratio of ERK1 phosphorylation to total ERK1. The mean value of the control group was set at 100, and the data for pups from MIA-injected dams were recorded as a percentage of the mean value of pups from saline-treated mothers. (C) Statistical analysis of the ratio of ERK2 phosphorylation to total ERK2. The mean value of the control group was set at 100, and the data for pups from MIA-injected dams were recorded as a percentage of the mean value of pups from saline-treated mothers; n = 6 for each group. Data are means ± SEM. *P＜0.05, **P＜0.01 (two-tailed Student's t-test).

Fig. 3. Effect of MIA on phosphorylation of ERK1/2 in the HIP of rat offspring. (A) Representative images of ERK1/2 phosphorylation in adolescent and adult offspring of rat dams intravenously injected with poly I:C (10 mg/kg) or saline on E9.5. (B) Statistical analysis of the ratio of ERK1 phosphorylation to total ERK1. The mean value of the control group was set at 100, and the data for pups from MIA-injected dams were recorded as a percentage of the mean value of pups from saline-treated mothers. (C) Statistical analysis of the ratios of ERK2 phosphorylation to total ERK2. The mean value of the control group was set at 100, and the data for pups from MIA-injected dams were recorded as a percentage of the mean value of pups from saline-treated mothers; n = 6 for each group. Data are means ± SEM. *P＜0.05 (two-tailed Student's t-test).

Fig. 4. Effect of MIA on neurofilaments in the PFC of rat offspring. (A) Representative images of NFM expression on PND40 and PND60 in offspring from rat dams intravenously injected with poly I:C (10 mg/kg) or saline on E9.5. (B) Statistical analysis of NFM expression. The mean value of the control group was set at 100, and the data for pups from MIA-injected dams were recorded as a percentage of the mean value of pups from salinetreated animals. (C) Representative images of NFH expression on PND40 and PND60 for offspring of rat dams intravenously injected with poly I:C (10 mg/kg) or saline on E9.5. (D) Statistical analysis of NFH expression; n = 6 for each group. Data are means ± SEM. *P＜ 0.05.

Fig. 5. Effect of MIA on neurofilaments in the HIP of rat offspring. (A) Representative images of NFM expression on PND40 and PND60 for offspring of rat dams intravenously injected with poly I:C (10 mg/kg) or saline on E9.5. (B) Statistical analysis of NFM expression. The mean value of the control group was set at 100, and the data for pups from MIA-injected dams were recorded as a percentage of the mean value of pups from salinetreated animals. (C) Representative images of NFH expression on PND40 and PND60 for offspring of rat dams intravenously injected with poly I:C (10 mg/kg) or saline on E9.5. (D) Statistical analysis of NFH expression; n = 6 for each group. Data are means ± SEM. *P＜ 0.05.

Fig. 6. Immunofluorescence studies. (A and B) Immunofluorescent staining of p-ERK (red) and DAPI (blue) in PFC (A) and HIP (B) of rats on PND40 and PND60 after early prenatal treatment with vehicle or Poly I: C. (C and D) Immunofluorescent staining for NFM and NFH in PFC (C) and HIP (D) of rats at PND40 and PND60 after early prenatal treatment with vehicle or poly I:C. 4. Discussion Schizophrenia is a complex psychotic disorder with multiple pathogenic etiologies. A better understanding of the disorder and the development of new treatment targets are essential. The ERK signaling pathway is known to be closely associated with the synaptic plasticity involved in neurogenesis and neuronal differentiation. The ERK signaling pathway may be the underlying basis for the cognitive deficits associated with schizophrenia30,31. ERK has a

key function in neuronal cells but its involvement in schizophrenia is poorly understood, although pharmacological studies suggest the ERK cascade to be important in schizophrenia development18,19,32. The study described herein was designed to explore ERK alterations in a rat model of schizophrenia and the furhter study is needed to verify the relationship between ERK expression and cognitive defects. Accumulating evidence associates prenatal infection with neurodevelopmental disorders, including schizophrenia and autism33. Animal models of MIA using the viral mimic poly I:C or other immune stimuli demonstrate behavioral impairments in offspring including anxiety, memory, and sensorimotor gating that are reminiscent of schizophrenic symptoms 34,35. In line with previous findings, our study found impaired spatial memory in the Y maze and abnormal PPI in adult offspring after maternal poly I:C challenge on E9.5 36. Therefore, this model can be used to assess the molecular mechanisms of cognitive impairment in schizophrenia. ERK1/2, a member of a major subfamily of mitogen-activated protein kinases, belongs to a class of serine/threonine protein kinases that regulate proliferation, differentiation, and survival of neuronal cells. Disruption of ERK-mediated signal transduction contributes significantly to the pathogenesis of various neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and many others 37. There is a widely accepted hypothesis that ERK1/2 opposes cell death38. Several lines of clinical evidence have shown abnormal activity of ERK signaling pathways in the postmortem brains of patients with schizophrenia39,40. In this study, we found a significant decrease in the activation of ERK in the HIP and the PFC of rat offspring after maternal poly I:C exposure in comparison to control animals. This finding is consistent with antipsychotic drug studies that have shown both clozapine and olanzapine injection in rats and mice to upregulate ERK1/2 signaling in brain regions18,19. The role for ERK in synaptic plasticity and memory is well documented 30,32. It is known that activation of ERK facilitates long-term potentiation and promotion of cell survival and

enhanced memory, while decreased p-ERK can impair long-term potentiation31. HIPdependent associative learning has been associated with the activated ERK/cAMP response element binding protein signaling pathway41. Runyan and Dash have shown that the ERK pathway modulates cognition in the rat PFC42 and that ERK1/2 activation can improve cognitive deficits43,44. Training in novel object recognition or in fear conditioning result in ERK activation in the HIP 5 min after the start of training, suggesting a role for ERK in the HIP following a learning episode45. We present evidence here that maternal poly I:C challenge results in decreased ERK activation in MIA offspring, which may be responsible for the cognitive impairments observed in MIA offspring. An interesting question is how decreased ERK1/2 phosphorylation in the PFC and the HIP is induced in MIA offspring? It is well-known that MIA can induce complex physiologic and biological changes in offspring that include inflammation, various receptor deficits, and enhanced neurotransmitter release46-48. In fact, MIA is known to increase dopamine release in rats, an effect which may lead to alteration of ERKs42. A decrease in ERK phosphorylation ratio may be due to an alterations in its upstream pathways 49. For example, erythropoietinproducing hepatocellular (Eph) B receptor signaling, implicated in dendritic spine development, has been reported to modulate the ERK signal transduction pathway50. Pathway dysfunction is involved in the phosphorylation of ERKs including the cascade of cAMPdependent protein kinase, which has been reported in schizophrenia, and may be responsible for decreased phosphorylation of ERKs51. ERKs may be a type of kinase that regulates neurofilaments 25,29,52. While little evidence exists for such a link in mammalian neurons, in vitro experiments with different cell lines, including N2a

25

and PC12

27

, have suggested such a linkage. Neurofilament expression and a

regulatory role for ERKs in neurofilament expression have not been previously examined in a MIA schizophrenia rat model. Herein we demonstrated abnormalities in NFM expression. However, NFH expression was apparently not related to ERK activity. Specifically, MIA caused a significant increase in NFH levels in the PFC at PND40, when there was no

significant alteration in pERKs/tERKs. Discrepancies in the expression of ERK1/2 and NFH suggest the possible existence of other signal transduction pathways regulating neurofilaments. Neurofilament proteins are the major cytoskeletal components of neurons, which determine neuronal morphology and are involved in structural plasticity as well as signal transduction. Neurofilament protein is reliably measured in cerebrospinal fluid and blood and is a marker of axonal damage in a variety of disorders, including multiple sclerosis and Alzheimer’s disease53-56. Axonal damage disrupts neural circuits and may play a role in cognitive impairment 56. Many studies have demonstrated elevated CSF NFL levels to be associated with faster cognitive decline56,57. Previous reports have shown altered protein levels for NFH, NFL, and NFM in schizophrenia58-62. Further, NFM and NFL have been shown to impact synaptic plasticity in schizophrenia63,64. Here we described for the first time an alteration in NFH and NFM protein levels in a MIA rat model, which simulates many clinical symptoms of schizophrenia, including cognitive impairment. With regard to axonal damage and synaptic plasticity, dysregulation of neurofilaments in MIA offspring may result in the cognitive deficits observed in the model and possibly in schizophrenia. This study has limitations. Although we found reduced activation of ERK and impaired memory in MIA rats, we cannot assume that these results are closely related, even though activation of ERK has been shown to enhance memory. Therefore, MIA-induced decrements in ERK activation within the HIP of offspring may not be directly associated with memory impairment in adult rats. It is important to note that abnormal expression of ERK proteins in the hippocampus was observed earlier than cognitive defects. Thus, the relationship between ERK expression and cognitive defects will require further exploration.

5. Conclusion

Our study has shown the potential involvement of ERKs in the pathogenesis of schizophrenia. Progress has been made in the development of therapeutic molecules for targeting of ERK

signaling. Therefore, improved understanding of the molecular mechanisms underlying regulation of ERK networks will be important in understanding the pathogenesis of schizophrenia. Further research will be required to determine if ERK pathway targeting can modulate the progression of schizophrenia. Acknowledgements We are grateful to LXL and WQL who designed the study and wrote the protocol. XS and YQH established the MIA animal model and wrote the manuscript. We would also like to thank FPS and XGG who helped in sample preparation and interpretation of the results. MS, MLS, WYG, LWZ, YFY, YZ, MLS, and MS undertook the statistical analysis and helped with molecular biological techniques. We thank all authors for their valuable assistance. Funding

This work was supported by National Natural Science Foundation of China (81671330 to LL); National Key Research and Development Program of China (2016YFC0904301, 2016YFC1307001); Medical Science and Technology Research Project of Henan Province (2018020371 to XS, 201702131 to YY); High Scientific and Technological Research Fund of Xinxiang Medical University (2017ZDCG-04 to LL); the training plan for young excellent teachers in Colleges and Universities of Henan (2016GGJS-106 to WL); the Science and Technology Project of Henan Province (192102310086 to WL; 162102310488 to WG); the Open Fund of Henan Key lab of Biological Psychiatry (ZDSYS2018008 to XS, ZDSYS2014002 to WG); the support project for the Disciplinary Group of Psychiatry and Neuroscience, Xinxiang Medical University. Declarations of interest

The authors declare no competing interest.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding authors upon reasonable request. Consent for publication Not applicable. Ethics approval and consent to participate All experimental protocols and procedures were approved by the Animal Care and Use Committee of the Henan Key Lab of Biological Psychiatry, Xinxiang, China. References 1 2

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