Background
Autism spectrum disorders (ASD) are defined by the disruption of language and social function and self-stimulatory or repetitive behaviors. The etiologic heterogeneity of ASD has complicated progress towards understanding the biological mechanisms of autism and developing targeted therapies. Recently, however, advances in autism genetics and progress in the study of animal models have provided evidence to suggest that some intracellular pathways are commonly affected in autistic patients, particularly the mitogen-activated protein kinase (
MAPK)/extracellular signal-related kinase (ERK) pathway [
1]. Genome-wide association studies [
2] and genomic copy number variant (CNV) analyses have identified enrichment in gene-sets involved in RAS (Rat Sarcoma)/
MAPK signaling and kinase activation in ASD individuals [
3]. Interestingly, a 593-kb deletion in chromosome 16p11.2, one of the most common CNVs associated with autism, contains the
MAPK3 (ERK1) gene [
4,
5]. In addition, a number of single-gene mutations implicated in syndromes such as “RASopathy” disorders [
1], fragile X syndrome [
6], and tuberous sclerosis [
7,
8], are also associated with disruption of the RAS/
MAPK signaling pathway and lead to pleiotropic neurocognitive impairments, including ASD [
1]. Furthermore, in mice, inactivation of
Mapk(Erk2) in the forebrain results in alterations in behavior that have similarities with those seen in autism [
9]. These findings provide preliminary evidence that regulation of ERK signaling may be broadly altered in autism.
The RAS/
MAPK signaling pathway mediates the transmission of signals from cell surface receptors to cytoplasmic and nuclear effectors. Diverse groups of molecular adaptors bind RAS and/or RAP1 (RAS-related protein 1) and initiate downstream signaling through ERK [
1]. Depending on enzyme kinetics, and sub-cellular distribution of each component, this pathway will mediate diverse cellular functions including proliferation, migration, differentiation, and cell survival [
10]. In the nervous system, this pathway is additionally involved in a diverse array of activity-dependent neuronal events, including synaptic plasticity, long-term potentiation or depression (LTP and LTD), and memory formation [
9,
11]. For example, in the transgenic mouse model of tuberous sclerosis, dysregulated ERK leads to impaired LTD, which was shown to mediate social behavioral deficits [
7].
Recently, the inbred mouse strain BTBR T + tf/J (BTBR) has been studied as a possible preclinical model of autism [
12], and we have recently shown that these behaviors are quantitatively linked to genetic loci [
13]. A total of six quantitative trait loci, meeting genome-wide significance for three autism relevant behaviors in BTBR, were identified on chromosomes 1, 3, 9, 10, 12, and X. Moreover, in a recently published biochemical evaluation of BTBR mice, the authors identified upregulation of the ERK signaling pathway in the newborn mice, suggesting, but not demonstrating directly, that this elevation in Erk activation was linked with the autistic behavior in BTBR [
14]. In this current study we evaluated the Ras
/ Mapk signaling pathway in BTBR mice, and tested whether there was a correlation between the degree of activation of the Ras/
Mapk pathways and autism-relevant traits, by testing intercrossed mice that all share varying degrees of the BTBR genome, allowing a stratified comparison of Erk activation and behavior. We also assessed whether levels of Erk activation in the brain correlated with levels of Erk activation in lymphocytes from the same animal. Both these lines of investigation provide preliminary evidence for the role of ERK activation in models of ASD and the capacity to monitor this activation in peripheral tissue.
Methods
Mice
BTBR, CD1 and C57BL/6 J (B6) lines were sourced from the Jackson Laboratory (Bar Harbor, Maine, United States). Mice from the same strain were bred either on site at the University of California, San Francisco or at The University of Queensland, under ethics approval from the respective University Animal Ethics Committees. Mice were weaned at P (postnatal day) 20 to 23 and then group-housed by sex in standard mouse cages containing two to four mice, following standard protocol. The day of vaginal plug was designated as E (embryonic day) 0 and the day of delivery as P0. BTBR and B6 were also bred and a total of 410 F2 mice were generated for behavioral testing over a period of two years in the laboratory of Dr Jacqueline Crawley at the NIMH (National Institute of Mental Health) in Bethesda, Maryland. The first cohort of 204 mice was generated by crossing F1 males and females derived from BTBR female and B6 male matings. The second cohort of 206 mice was generated by the reciprocal cross of F1 males and females derived from B6 female and BTBR male matings. This ensured equal representation of the BTBR and B6 X chromosomes in the final F2 cohort. Further details have been described previously [
13].
Autism-relevant behaviors evaluated in F2 mice
Juvenile reciprocal social interactions were assessed on P21. The test mouse and age- and sex-matched B6 control mice were simultaneously placed in the field and their interactions were videotaped for 10 minutes. Social behaviors including nose-to-nose sniff (sniffing the nose and snout region of the partner), front approach (moving towards the partner, in a head-on manner), and push-crawl (pushing the head underneath the partner’s body or squeezing between the wall or floor and the partner, and crawling over or under the partner’s body, combined as a single parameter) were evaluated and scored by a highly trained observer, using the Noldus Observer 5.0 software (Noldus Information Technology Wageningen, Netherlands).
Social approach was assayed in automated three-chambered apparatus between eight and 12 weeks of age. The test mouse was briefly confined to the center chamber while the clean novel object was placed in one of the side chambers. A novel mouse, previously habituated to the enclosure, was placed in an identical wire cup located in the other side chamber. The side containing the novel object and the novel mouse alternated between the left and right chambers across subjects. After both stimuli were positioned, the two side doors were simultaneously lifted and the subject was allowed access to all three chambers for 10 minutes. Number of entries and time spent in each of the three chambers were automatically detected by photocells embedded in the doorways and tallied by the software. In addition, time spent sniffing the novel mouse was scored by human observers [
13].
P0 and P30 BTBR and B6 mice were euthanized by administration of inhaled CO2 followed by cervical dislocation. Whole brains were extracted and homogenized in ice-cold NP-40 lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 20 mM MgCl2, and 0.5% NP-40 with the addition of protein phosphatase inhibitor (PhosSTOP, Product number: 04906845001, Roche Diagnostics, Indianapolis, USA) and protease inhibitor cocktails (Complete Protease Inhibitor, Product number: 11697498001, Roche Diagnostics, Indianapolis, United states). After centrifugation (13,000 x-g, 15 minutes, 4°C), the supernatants were collected. The protein concentration was measured using the Bio-Rad protein assay (Life Science, Hercules, CA, USA) and all protein extracts were stored at −80°C.
Three-month-old F2 offspring from a cross of BTBR and B6 mice were anesthetized with ketamine and xylazine then perfused with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde via cardiac puncture, and the brains were extracted. One hemisphere was used for anatomic evaluation and proteins were isolated from the prefrontal cortex and cerebellum of the other hemisphere using the Qproteome FFPE kit (Qiagen, Hilden, Germany) [
15].
To extract protein from lymphocytes, the spleens of the mice were removed and mechanically meshed through a cell strainer. Cells were washed with PBS and then 2 to 3 μl of red blood cell (RBC) lysis buffer (Biolegend, San Diego, USA) was added to the cell aliquot for 2 minutes to lyse the RBCs, and the remaining lymphocytes were pelleted and washed. Subsequently, NP-40 lysis buffer was added to the cell aliquot and the cellular extract subjected to centrifugation. The extracted protein supernatant was stored at −80°C.
Protein electrophoresis and Western blot analysis
Samples were denatured in sample buffer by heating at 95°C for 5 minutes. A total of 20 micrograms of protein per lane was run on a 12% acrylamide gel for 2 hours at 100 V. The proteins were transferred to a PVDF membrane at 50 V for 1 hour and the PVDF membrane ( Bio-RAD, Life Science Research, Hercules, USA) was then blocked for 1 hour with 5% dry milk in PBS with 0.1% Tween 20. Thereafter, the primary antibodies were applied overnight with continuous shaking at 4°C. The primary antibodies included rabbit mAb Ras, Phospho-mitogen-activated protein kinase kinase (MEK)1/2 (Ser217/221), rabbit mAb MEK1/2 (47E6), rabbit mAb Phospho-ERK (Thr 202/Tyr 204), p44/42 MAPK(ERK1/2), rabbit mAb β-actin (Cell Signaling). The blots were then washed and incubated with secondary antibody, HRP (Horseradish peroxidase) -linked anti-rabbit IgG, (Cell Signaling) for 1 hour at room temperature. Immunoreactive bands were visualized by using the enhanced chemiluminescence detection system (Pierce) and exposed to autoradiography film (HyBlot film CL). Densitometry was performed using ImageJ software, based on the standard protocol described via the National Institutes of Health (Bethesda, Maryland, United States). The intensity of the each protein is normalized to the actin signal obtained after stripping the same membrane and reprobing for actin in the same lane.
Immunohistochemical analysis of cellular proliferation
Pregnant BTBR and control CD1 dams (n ≥6) were given an intraperitoneal injection of 5-Ethynyl-2′-deoxyuridine (EdU, 10 μg per kg of body weight, Invitrogen, Life Technologies, Grand Island, NY, USA) 30 minutes prior to sacrifice. Dams were then anesthetized with ketamine and xylazine and embryos collected at E14 and E17. E14 heads were immersion fixed in 4% paraformaldehyde (4% PFA) w/v in PBS pH7.4 (ProSciTech, Kirwan Australia), whilst E17 embryos were transcardially perfused with saline (0.9% NaCl w/v in H2O) followed by 4% PFA. Brains were extracted and sectioned at 50 μm thickness on the coronal orientation using a vibratome. Representative sections of the rostral telencephalon were mounted, post-fixed with 4% PFA for 20 minutes, and subjected to sodium citrate antigen retrieval (125°C at 15 psi for 4 minutes in sodium citrate buffer, 10 mM C6H5Na3O7, 2H2O, and 0.05% v/v Tween 20 in MilliQTM H2O, at pH 6.0). Sections were blocked in normal goat serum (10%)/0.2% Triton-X (Sigma-Aldrich, St Louis, Missouri, United States) in PBS for 30 minutes. EdU detection was performed using a Click-IT EdU Alexa Fluor 488 Imaging Kit (Invitrogen, Life Technologies, Grand Island, NY, USA), according to the manufacturer’s instructions. The slides were then light protected and re-blocked in 10% normal goat serum/0.2% Triton-X 100 in PBS for 2 hours. The following primary antibodies were applied to the slides in the blocking solution and left overnight: rabbit anti-phospho-histone H3 (Ser 10, 1:500, EMD Millipore, Darmstadt, Germany), rabbit anti-Pax6 (1:500, EMD Millipore, Darmstadt, Germany), rabbit anti-TBR2 (1:500, Abcam, Cambridge, USA) mouse anti-human Ki67 (1:500, BD Biosciences, San Jose, USA). After PBS washes, sections were then incubated for 3 hours with Alexa Fluor 555 donkey anti-rabbit IgG (1:500, Invitrogen, Life Technologies, Grand Island, NY, USA)) secondary antibody. Biotinylated donkey anti-mouse IgG (1:500, Jackson ImmunoResearch, West Grove, USA) was then applied for 1 hour. Following further PBS washes, Alexa Fluor 647 Streptavidin conjugate (1:500, Invitrogen Life Technologies, Grand Island, NY, USA)) was applied for one hour. Slides were counter-stained with nuclear marker DAPI (diamidino-2-phenylindole) (1:1000, Invitrogen, Life Technologies, Grand Island, NY, USA), washed and cover-slipped with ProLong Gold (Invitrogen Life Technologies, Grand Island, NY, USA). Images were obtained at 20x magnification in a single representative z-plane using an inverted spinning disk confocal microscope equipped with Hamamatsu Flash4.0 scientific CMOS camera (Hamamatsu, Japan) and acquired with Slidebook (3i Intelligent Imaging Innovations, Denver, USA). Images were pseudo-colored for presentation in Adobe Photoshop (Adobe Systems Incorporated, San Jose, United States).
Quantification of cellular proliferation
Cell counts were performed on a region of interest from the neocortex, midline, and ganglionic eminence automatically using the spot analysis module in Imaris (Bitplane, Zurich, Switzerland). An appropriate threshold was set for detection of positive cells for each marker but kept constant between control and BTBR groups. Absolute cell counts were imported into Prism v.6 (GraphPad, La Jolla, USA) and presented as mean ± standard error. Statistical significance was determined at P <0.05, using the Mann-Whitney U test.
Statistical analysis
All data are shown as mean ± SEM. Group comparisons are analyzed using the non- parametric Mann-Whitney U test, using GraphPad Prism v.6 (P <0.05 was considered statistically significant). Non-parametric correlation (Spearman correlation) was also conducted to compare paired measurements of Mapk/Erk levels in the brain and lymphocytes.
Discussion
In this study, we observed increased activation of the Ras/Erk pathway in the brains of BTBR mice, a strain that has social and behavioral deficits that may have relevance to ASDs [
13]. Increased p-Erk levels in the newborn BTBR strain are consistent with a prior study that evaluated this pathway in BTBR mice [
14]. However, this increased activation of Erk was less pronounced in adolescent BTBR mice. We also showed that total protein levels of Erk in the brains of BTBR mice are not increased compared to B6 mice. Thus, we hypothesize that the possible contribution of the
Mapk/Erk signaling pathway to neurocognitive impairment in this mouse model of autism occurs through posttranslational modification of the Erk pathway. In support of this hypothesis, we showed that activation of the Erk signaling pathway, but not total Erk protein levels in prefrontal cortex, is associated with the degree of social impairment in intercrossed F2 mice with a mixed genetic background.
How might a change in the levels of ERK activation lead to behavioral abnormalities? Increased activation levels of
MAPK/ERK in neurons have been shown to selectively increase pools of mRNAs encoding adhesion molecules and scaffolding proteins [
22,
23]. Increased expression levels of these molecules can change the balance between excitatory and inhibitory synapses [
23], which has been suggested as a likely basis for impaired cognition and possibly for ASD [
24,
25]. In support of these hypotheses, Seese
et al. showed a negative correlation between the intensity of synaptic p-Erk1/2 immunolabeling and cognitive function across BTBR mice [
26]. This is clearly a complex issue, as the RAS/ERK signaling pathway is embedded in a network of other signaling pathways. ERK1/2 also has more than 70 different cytoplasmic and nuclear substrates, regulating many fundamental cellular processes. It is possible that each of the different proteins in this network has a cumulative effect on cognitive and behavioral impairments. However, more focused experiments are required to evaluate the role of each kinase individually. In contrast to the prefrontal cortex, when we evaluated the cerebellum of F2 mice, p-Erk levels were similar between mice with high and low social behavior scores, suggesting that the cerebellum may not be a focus of behavioral changes in this mouse strain. However, because we were only able to evaluate a limited number of F2 mice, we cannot rule out alpha or beta errors in our preliminary findings.
Because the
MAPK/ERK pathway activation has been shown in many cell types to be central for cell division and differentiation, we evaluated whether BTBR mice had, in addition to elevated p-Erk, changes in cell proliferation and apoptosis in the developing forebrain. Evaluation of these parameters during a critical window of cerebral cortex development revealed distinct spatio-temporal changes in proliferation, but not apoptosis. Decreased cellular proliferation in the cerebral cortex throughout this developmental period is consistent with other studies that reported a robust reduction in neurogenesis in adult BTBR mice [
27], which also lead to decreased brain volume and cerebral white matter in this strain [
28]. To explore the role of
Mapk/Erk on neural cell proliferation, Yang
et al. overexpressed c-Raf in cultured cortical neurons. They observed impairment in neuronal cell differentiation and maturation and unchanged apoptosis in the presence of amplified
Mapk/Erk, which is consistent with our findings [
19]. However, the role of the
Mapk/Erk pathway in regulating neuronal cell division and maturation is complex, as other studies have demonstrated that decreased
Mapk/Erk activation can also disrupt progenitor proliferation [
29‐
31]. Indeed, it is possible that any change from a homeostatic balance in this pathway may alter cognition and behavior. However, we were unable in this study to directly compare proliferation with behavior, so it is still remains possible that this change represents strain-to-strain variation without correlation with behavior.
In addition to these findings in the developing brain, we also found that the degree of activation of the Erk pathway directly correlated when comparing brain tissue to lymphocytes, suggesting that biological correlates of social impairment could be measured in peripheral blood. Based on these findings in a mouse strain, we speculate that this pathway could be dysregulated in the lymphocytes of autistic patients. Further investigation will be required to assay the levels of MAPK/ERK in the lymphocytes of individuals with autism and whether this could serve as a framework for developing a potential biomarker for autism.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AF participated in the design of the study, carried out the protein assays, performed the statistical analysis and contributed in drafting the manuscript. DJD participated in generating F2 mice, analyzing the behavioral testing and parafinizing the mice. ER participated in generating F2 mice, analyzing the behavioral testing and parafinizing the mice. JL contributed to the protein assays. IG participated in the immunohistochemical assay and its analysis, prepared the figures and contributed to writing the paper. LM participated in the immunohistochemical assay and its analysis. LJR participated in designing the immunohistochemical assay, mentoring the experiments and editing the manuscript. SS participated in the design of the study and performed the statistical analyses. EHS was the principal investigator who conceived of the study, supervised all the experiments and drafted the manuscript. All authors read and approved the final manuscript.