Elevated levels of cortisol, brain-derived neurotropic factor and tissue plasminogen activator in male children with autism spectrum disorder

Hasan Bozkurt1 | S¸eref S¸ims¸ek2 | Serkan S¸ahin1

1Department of Child and Adolescent Psychiatry, Gaziosmanpasa University School of Medicine, Tokat, Turkey
2Department of Child and Adolescent Psychiatry, Dicle University School of Medicine, Diyarbakır, Turkey

Hasan Bozkurt, Department of Child and Adolescent Psychiatry, Gaziosmanpasa Hospital, Yeniyuzyil University School of Medicine, Çukurçes¸me Street, No: 51, 34245
I_stanbul, Turkey.
Email: [email protected]

Several studies demonstrated biological effects of cortisol, brain-derived neuro- trophic factor (BDNF) and tissue plasminogen activator (tPA) on human metabo- lism and central nervous system. Our study investigated the serum levels of tPA along with BDNF and cortisol in children with autism spectrum disorder (ASD). Thirty three male children with ASD ranging in age from 2 to 15 years were selected for the study group and 27 age-matched healthy male children were selected for the control group. The ASD severity was determined by the score on the Autism Behavior Checklist (ABC). The mean cortisol levels for the study group and the control group were 79.1 30.2 ng/ml and 60.0 25.1 ng/ml, respectively. The mean BDNF levels for the study group and the control group were 5.9 2.8 ng/ml and 3.7 1.8 ng/ml, respectively. The mean tPA levels for the study group and the control group were 32.9 18.5 ng/ml and 25.5 15.1 ng/ml, respectively. Cortisol, BDNF and tPA levels were significantly higher in the study group compared to the control group (p < 0.001). There was no statistically significant effect in terms of age, ABC total and subscale scores on serum cortisol, BDNF and tPA levels in the study group (p > 0.05). It may be suggested that elevations may indicate a role in the pathogenesis of ASD or it may be the case that ASD may alter the levels or pathways of these metabolic factors.

Lay Summary
The underlying mechanism or a specific metabolic target relevant to autism spec- trum disorder (ASD) has not yet been identified. Cortisol, brain-derived neuro- trophic factor (BDNF) and tissue plasminogen activator (tPA) have biological effects on neuroplasticity but little is known about the role of cortisol and tPA- BDNF pathway in ASD. In the present study focused on male children with ASD, we have found higher blood levels of cortisol, BDNF and tPA than their healthy peers. This is the first clinical study to evaluate the serum tPA levels along with BDNF and cortisol in ASD. The results suggest that several neurotrophic and other related markers should be born in mind while examining children with ASD.

autism, brain-derived neurotrophic factor, cortisol, tissue plasminogen activator

Autism spectrum disorder (ASD) is a neu- rodevelopmental disorder characterized by persistent def- icits in social communication and social interaction with restricted, repetitive patterns of behavior, interests, or activities (APA, 2013). The etiology of ASD remains unknown with many factors including genetic, environmental, prenatal and perinatal involvement, and neuroanatomical abnormalities. Megalancephalic brain, neuronal disorganization and abnormal migration, corti- cal heteropia, age-dependent pruning defect, reduction and incursion in purkinje cells and sometimes gliosis have been suggested to be the neuropathological hallmarks of ASD (Volkmar & Pauls, 2003). However, research on the pathogenesis of ASD has been ongoing for about half a century and the etiology still remains unknown.
Cortisol, the primary glucocorticoid in humans, is released from the adrenal cortices of the hypothalamic pituitary adrenal (HPA) axis. It has a variety of effects on cardiovascular function, immunity, metabolism, and neurobiology with being involved in several vital biologi- cal processes and interactions (Herman & Cullinan, 1997). Cortisol is also central to the physiological response to physical or perceived psychological stress. The diurnal rhythm and responsiveness of cortisol has been evaluated in many studies suggesting greater circa- dian dysregulation in ASD relative to typically developed (TD) healthy controls (Corbett et al., 2006, 2008; Edmiston et al., 2017; Hadwin et al., 2019; Muscatello & Corbett, 2018; Spratt et al., 2012).
Brain-derived neurotrophic factor (BDNF), a mem- ber of the neurotrophin family of survival-promoting molecules, plays a vital role in the growth, development, maintenance, and function of several neuronal systems (Huang & Reichardt, 2001). BDNF is involved in the survival and differentiation of dopaminergic neurons in the developing brain and plays an important role in the formation and the plasticity of synaptic connections (Binder & Scharfman, 2004). It is also trophic for seroto- nergic neurons, and abnormalities in serotonin levels are among the most common biochemical findings in ASD (Pardo & Eberhart, 2007). However, the importance of circulating BDNF in ASD is still unclear because several recent studies demonstrate increased systemic levels of BDNF in children with ASD, while other studies report no differences or even decreased levels (Francis et al., 2018; Mansour et al., 2010; Meng et al., 2017; Nel- son et al., 2006; Ricci et al., 2013; Taurines et al., 2014; Zhang et al., 2014; Zheng et al., 2016).
BDNF arises from a precursor, proBDNF, which is cleaved to produce the mature protein through the tissue plasminogen activator (tPA) pathway, and represents one mechanism that can regulate the action of BDNF. tPA is a serine protease produced by endothelial cells and converts plasminogen to active plasmin which breaks down fibrin clots (Collen & Lijnen, 1991). This enzyme has been shown to be produced by neurons and glia in the central nervous system and to play physiolog- ically and pathologically crucial roles. It is highly expressed in the adult rodent brain regions including amygdala, hippocampus, and cerebellum (Pawlak et al., 2003). Neuroanatomical and functional abnor- malities related to those areas have been demonstrated in children with ASD. In this context, tPA has been also reported to modulate emotion in a social context in rodents (Nakamura et al., 2015).
There are studies associated with the specific markers in ASD including cortisol with HPA axis and BDNF in the literature. However, to the best of our knowledge, only one study has been conducted investigating the serum tPA levels in children with ASD (Simsek et al., 2016). Biological interactions between cortisol, BDNF and tPA have been shown in several studies. Glu- cocorticoids have been found to play a role in the regula- tion of BDNF inducing gene expression and altering its levels (Jeanneteau & Chao, 2013). tPA has been also demonstrated to contribute to long term potentiation of neurons leading to changes in the proBDNF/BDNF ratio (Nagappan et al., 2009). Considering such interactions involved in many processes related to neurodevelopment and neuroplasticity, it becomes crucial to examine the serum levels of these three markers in ASD. They may play a role in the pathophysiology of ASD or ASD may also alter the levels of these markers. Therefore, we aimed to assess whether cortisol, BDNF and tPA levels differ in children with ASD from their TD peers in this study.


Thirty three male children with ASD ranging in age from 2 to 15 years were selected for the study group and 27 age-matched healthy male children were selected for the control group. Subjects in the study group were recruited among male children and adolescents who were referred to Department of Child and Adolescent Psychiatry in two centers during a period of six months. The cases considered as ASD were enrolled consecu- tively in the study, and the psychiatric interviews were conducted by two expert psychiatrists within 6-month intervals. Because serum tPA levels interact with estro- gen and progesterone, we did not include female patients with ASD.
Subjects with any genetic syndrome (e.g., Down syn- drome, fragile X, Rett syndrome) and any medical dis- order (e.g., epilepsy, clinically active infection, Cushing syndrome, and morbid obesity) and with a history of past or current cortisol therapy or vitamins were excluded from the study group. Healthy male children, living in similar addresses with patients, similar in age, and having no history of psychiatric illnesses were selected as healthy control group. The exclusion criteria for the control group were neurodevelopmental disor- ders (e.g., ASD, intellectual disability, communication disorders), presence of any neurological disorder, clini- cally active infection, and having a history of past or current cortisol therapy or vitamins. Written informed consent was obtained from parents and the study proto- col was approved by the Non-invasive Clinical Research Ethics Committee of Gaziosmanpasa University.


Autism behavior checklist (ABC)
ABC was developed by Krug et al. (1980). It has been used to evaluate the severity of autism symptoms. ABC consists of five subscales which have a 57-item scale including sensory, relationship building, the use of the body and objects, language skills, social and self-care skills. The lowest score of the scale is 0 and the highest score is 159. The scale has been adapted to Turkish by Irmak et al. (2007).

Biochemical analysis
Blood samples were obtained in the morning between 09:00 and 10:00 h. The samples were collected in gel tubes. After withdrawal, blood samples were allowed to rest for 15 min for clotting. Then, blood samples were centrifuged at 5000 rpm for 6 min. The sera were transferred to 1.5 ml polypropylene tubes and stored at 80◦C until the analysis. Cortisol, BDNF and tPA levels were measured using a human ELISA Kit (Sunred Biotech Co., Ltd, China and Shanghai LZ Biotech Co., Ltd).

Firstly the diagnosis process of ASD was conducted in referred subjects. Two expert child and adolescent psychi- atrists examined the subjects consecutively and diagnosed them as ASD according to Diagnostic and Statistical Manual of Mental Disorders, 5th edition (APA, 2013). Patients with given the same diagnoses by two experts were included and cases without diagnostic consensus were excluded. The severity of autistic symptoms was assessed with the ABC scale. Children with ASD having a loss of language and social skills between 18–30 months were also diagnosed with regressive type of autism. Hearing tests were applied to all participants. Blood draw pro- cesses were performed by experienced pediatric nurses and phlebotomists. A parent was present by holding the child during the venipuncture to reduce psychological stress. Another phlebotomist helped for assistance if the parent had difficulties to apply full instructions on how to comfort the child. Parents were also told to make their children have breakfast in the morning before applying to hospital for phlebotomy. Blood samples of children were collected regardless of the time they wake up between 9 and 10 a.m. once a day. ELISA was used to assay serum levels of cortisol, BDNF, and tPA. To mini- mize assay variance, all measurements were conducted on the same day. All experiments were performed in duplicate. The tests were performed according to the manufacturer’s instructions.

Statistical analysis
The student’s t-test was used to compare normally dis- tributed variables in independent groups, and the Mann– Whitney U test was used to compare nonparametric or ordinal variables. The effects of age and ABC scores on serum levels were evaluated using a two-way ANOVA and ANCOVA tests. Pearson’s test was used to evaluate correlation coefficients and statistical significance of normally distributed variables, and Spearman’s test was used to evaluate non-normally distributed variables. The values were given as mean standard deviation (SD). A value of p < 0.05 was considered statistically significant. RESULTS The study group consisted of 33 male children with a mean age of 47.7 32.0 months and the control group consisted of 27 healthy male children with a mean age of 47.2 12.1 months. Mean number of siblings in the study and control groups were 2.4 1.3 and 2.6 1.6, respectively. 24.2% of parents in the study group were found to have consanguineous marriages (all cousin types) while 25.9% of parents in the control group had consanguineous marriages (all cousin types). There was no significant difference between the groups in terms of mean age of the participants, age of the mother or father, number of siblings and the rates of consanguineous mar- riage (p > 0.05). Table 1 shows the socio-demographic attributes of the study and control groups.
The mean cortisol levels for the study group and the control group were 79.1 30.2 ng/ml and 60.0 25.1 ng/ml, respectively. The mean BDNF levels for the

TABLE 1 Socio-demographic variables of the study and control groups
Study group (n = 33) Control group (n = 27) t value p value
Age (months) 47.7 32.0 47.2 12.1 0.086 0.93
Mother’s age (years) 29.6 5.8 32.9 6.2 —2.050 0.05
Father’s age (years) 34.5 6.5 36.5 6.5 —1.161 0.25
Number of siblings 2.4 1.3 2.6 1.6 —0.533 0.59
Consanguineous marriage (%) 24.2 25.9 1.00

study group and the control group were 5.9 2.8 ng/ml and 3.7 1.8 ng/ml, respectively. The mean tPA levels for the study group and the control group were 32.9 18.5 ng/ml and 25.5 15.1 ng/ml, respectively. Cortisol, BDNF and tPA levels were significantly higher in the study group compared to the control group (p < 0.05). The biochemical parameters in the study and control groups are displayed in Table 2. The mean total ABC score was 80.2 20.0 in the study group. The ABC subscale scores were found to be 10.1 4.5 for sensory, 20.4 5.1 for relating, 18.8 5.5 for body and object use, 18.7 5.0 for language, and 12.8 3.6 for social and self-help. Regressive type of autism was observed in 21.2% of the subjects with ASD (n = 7). The total ABC scores and language, social and self-help subscale scores were significantly higher in sub- jects with regressive autism than those without regression (p < 0.05). Two-way analysis of variance (ANOVA and ANCOVA) was conducted in order to assess the contri- bution of age and ABC total and subscale scores, regres- sive type of autism on serum cortisol, BDNF and tPA levels of the study group. There was no statistically signif- icant effect in terms of age, ABC total and subscale scores, and the regressive type of autism on serum corti- sol, BDNF and tPA levels in the study group (p > 0.05). Bivariate correlation also showed no statistically signifi- cant relationship between age and serum levels in the study and control groups (r = —0.146, p = 0.367, and r = —0.078, p = 0.631, respectively).

The increasing findings represent that several factors related to neuronal, endocrine and immune system play an important role in the pathophysiology of ASD. So it is crucial to examine the blood levels of markers associated with those systems in individuals with ASD whether they differ from non-ASD healthy individuals and also to determine whether these markers affect autism severity. So the present study focused on investigating the serum cortisol, BDNF, and tPA levels in children with ASD comparing with those of TD healthy controls. The results suggest that cortisol, BDNF, and tPA levels were signifi- cantly higher in children with ASD than their TD peers. To the best our knowledge, this is the first clinical study examining the serum tPA levels along with cortisol and BDNF in children with ASD.
Given the importance of cortisol as a marker involved in many regulatory processes including immune and met- abolic functioning, we have examined the serum cortisol levels. Children with ASD demonstrated significantly higher cortisol levels than healthy children in the present study. The normal circadian pattern of cortisol is a sharp increase in the morning hours, with a gradual decline throughout the day until it reaches its nadir during night- time sleep (Smyth et al., 1997); deviation from this pat- tern is suggestive of HPA-axis dysregulation. Some studies have focused on specific aspects of this pattern (e.g., cortisol awakening response, daily decline, variabil- ity) (Corbett & Schupp, 2014) while others examined cor- tisol levels once a day like our study (Curin et al., 2008). Using plasma cortisol collected in the morning hours, Curin et al. (2003) and Hamza et al. (2010) found lower cortisol levels in children with ASD relative to TD con- trols in contrast to our findings. However no difference has been reported in cortisol levels between ASD group and TD controls in other studies using the same method (Strous et al., 2005; Tani et al., 2005). Measuring cortisol levels in response to a stressful trigger (blood draw), Spratt et al. (2012) also found a significantly higher serum cortisol response in children with autism. Simi- larly, the blood draw in the current study may also have contributed to higher cortisol levels. Despite the inconsis- tent results, most of the studies suggest greater circadian dysregulation of cortisol in ASD groups relative to age- matched TD controls.
In view of the effect of BDNF in the development of neurons, it may be suggested that BDNF may play a role in the pathogenesis of autism. In the present study aimed at investigating the serum levels of BDNF, we found increased levels of BDNF in children with ASD com- pared with TD controls. This is consistent with other studies demonstrating elevated BDNF levels in patients with ASD (Bryn et al., 2015; Connolly et al., 2006; Cor- reia et al., 2010; Wang et al., 2015). There have been con- troversies in regard to the role of BDNF in the etiology of ASD. So in addition to studies showing higher levels of BDNF among autistic patients compared to controls, several studies also demonstrated reduced BDNF in ASD patients or no differences at all (Abdallah et al., 2013; Hashimoto et al., 2006; Nelson et al., 2006; Rodrigues et al., 2014). A meta-analysis of studies

TABLE 2 Biochemical parameters of the study and control groups
Study group (n = 33) Control group (n = 27) t value p value
Cortisol (ng/ml) 79.1 30.2 60.0 25.1 2.609 0.009*
tPA (ng/ml) 32.9 18.5 25.5 15.1 2.032 0.035*
BDNF (ng/ml) 5.9 2.8 3.7 1.8 3.762 <0.001* Abbreviations: BDNF, brain-derived neurotrophic factor; tPA, tissue plasminogen activator. *Statistically significant. measuring peripheral blood levels of BDNF in children with ASD also showed that neonates diagnosed with ASD later in life did not have altered blood BDNF levels, whereas children in the non-neonate ASD group manifested increased peripheral blood levels of BDNF (Qin et al., 2016). The present study with non-neonate ASD children is in line with the findings of this meta-analy- sis, however, longitudinal studies rather than cross- sectional are necessary for accurate outcomes. On the other hand differences in the assay and sample test methods, ana- lytic platforms used, and patient populations may explain the inconsistencies among studies (Croen et al., 2008). The increased BDNF response in ASD has been considered as an immune response which may be dysregulated in autism since BDNF is also produced by activated T-cells, B-cells, and monocytes (Ricci et al., 2013). However, more research is needed in order to clarify the role of BDNF in the pathophysiology of autism. Since tPA, a serine protease involved in pro-BDNF cleavage to BDNF, is among the least examined markers in ASD, we further investigated plasma levels of tPA in children with ASD comparing with that of healthy con- trol subjects in addition to cortisol and BDNF. We found increased tPA levels in children with ASD in this study. To the best of our knowledge, there is only one study examining the tPA levels in individuals with ASD in the literature (Simsek et al., 2016). Simsek et al. (2016) found higher tPA levels in male children with ASD than their TD peers like the present study. There is a dichotomy of tPA influence on the CNS. tPA has a role in neuro- protective or neurotoxic processes depending on the type of neurons, the type of stress paradigm and the level of t- PA (Chevilley et al., 2015). tPA has neurotoxic effects particularly on cortical neurons (Nicole et al., 2001) and Purkinje neurons (Cops et al., 2013; Li et al., 2013), but neuroprotective effects particularly on hippocampal neu- rons (Lemarchand et al., 2016). In an animal study, tPA has been shown to be involved in social behavior and tPA deficiency might elicit social anxiety suggesting its effect in modulating emotions (Nakamura et al., 2015). Increased tPA levels found in the present study seem to be inconsistent with this finding since ASD is associated with deficits in social communication and social interac- tion. However, another study found that tPA can inhibit NMDA receptors in hippocampal neurons and this effect requires tPA to be proteolytically active without involv- ing plasminogen as a substrate and such activity related to NMDA-mediated changes in synaptic plasticity can be an irreversible event (Robinson et al., 2015). Elevated tPA levels may also contribute to the increased levels of BDNF since the tPA/plasmin system is the most signifi- cant protease involved in the cleavage of proBDNF to BDNF (Pang et al., 2004). But more research is necessary to understand the exact mechanism of tPA in ASD. Furthermore, cortisol, BDNF and tPA levels of the study group were not significantly associated with age, ABC total and subscale scores, and the regressive type of autism in our study. In an earlier study conducted by Tordjman et al., no significant relationship has been reported between autism severity based on the autism diagnostic observation schedule (ADOS), and cortisol levels, like the present study (Tordjman et al., 1997). However, Hamza et al. (2010) reported that autism sever- ity, based on the childhood autism rating scale (CARS) score, was significantly and negatively correlated with basal and stimulated cortisol levels. On the other hand, Mansour et al. (2010) showed no correlation between BDNF and severity of autistic features assessed by CARS while Meng et al. (2017) indicated that levels of BDNF increased with severity of ASD by the CARS score. Wang et al. (2015) also suggested that BDNF levels may be associated independently with the severity of ASD, and higher BDNF levels could be considered an independent diagnostic marker of ASD since they found a significant positive association between serum BDNF levels and CARS scores in their study. A recent study by Barbosa et al. (2020) evaluated the value of BDNF level as a possible auxiliary marker for the diagnosis of ASD in this context. Age is an important factor on serum corti- sol levels in children and research attests to age-related changes in diurnal cortisol (Titman et al., 2020). In the only study investigating the correlation between tPA levels, age and ASD severity, Simsek et al. (2016) found that tPA level was correlated negatively with age in ASD and the severity of ASD was correlated positively with age. Despite the broad age range of ASD group, age was found to have no significant effect on serum hormone levels in the present study. Further studies are warranted to clarify the inconsistent results regarding the associa- tion between cortisol, BDNF, tPA levels and autism severity. Our study has several limitations that should be addressed. First is the cortisol sampling method since we collected plasma cortisol once a day between 09:00 and 10:00 h, which did not consider individual differences in waking nor the potential stress effect of phlebotomy. Another limitation is that we could investigate only BDNF not pro-BDNF since the ELISA method is not able to detect serum pro-BDNF levels. The structured psychiatric interview for the diagnosis of autism (ADI-R and ADOS) has not been applied due to lack of validity and reliability in our country. Considering only male patients, lack of the intellectual functioning assessment of the study group and low case numbers are the other limi- tations of the present study. Despite these limitations, this is the first clinical study to evaluate the serum tPA levels along with cortisol and BDNF in children with ASD. Differently from the exis- ting studies examining only one biological marker like cortisol or BDNF or tPA, the present study is unique for its assessing the serum levels of all these markers together in children with ASD. Since the complexity of ASD is considered to arise from multiple factors affecting the pathophysiologic features of the disease, our study examining biomarkers involved in various neurobiologi- cal areas has more importance. So it is also the first study in ASD population to do a multisystemic research includ- ing neuronal, endocrine and immune systems in this con- text. Increased serum levels of tPA, BDNF and cortisol were found in children with ASD compared to healthy controls. Since the prevalence of ASD is gradually increasing in recent years, several neurotrophic and other related markers should be born in mind while examining this population. However more research is needed to fur- ther explore the relationship between ASD and these biomarkers. 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