The efficacy and Tolerability of the clonidine Transdermal Patch in the Treatment for children with Tic Disorders: a Prospective, Open, single-group, self-controlled study
February 2017

Jaime Kulisevsky
Pedro Chana
Pratap Chand
Yue Hu
Pan-Pan Song
Department of Neurology, Children's Hospital Affiliated to Chongqing
University of Medical Sciences
ChongqingChina

Ministry of Education Key Laboratory of Child Development and Disorders
ChongqingChina

Key Laboratory of Pediatrics in Chongqing
CSTC2009CA5002ChongqingChina

Chongqing International Science and Technology Cooperation Center for Child Development and Disorders
ChongqingChina

Li Jiang
Department of Neurology, Children's Hospital Affiliated to Chongqing
University of Medical Sciences
ChongqingChina

Ministry of Education Key Laboratory of Child Development and Disorders
ChongqingChina

Key Laboratory of Pediatrics in Chongqing
CSTC2009CA5002ChongqingChina

Chongqing International Science and Technology Cooperation Center for Child Development and Disorders
ChongqingChina

Xiu-Juan Li
Department of Neurology, Children's Hospital Affiliated to Chongqing
University of Medical Sciences
ChongqingChina

Ministry of Education Key Laboratory of Child Development and Disorders
ChongqingChina

Key Laboratory of Pediatrics in Chongqing
CSTC2009CA5002ChongqingChina

Chongqing International Science and Technology Cooperation Center for Child Development and Disorders
ChongqingChina

Si-Qi Hong
Department of Neurology, Children's Hospital Affiliated to Chongqing
University of Medical Sciences
ChongqingChina

Ministry of Education Key Laboratory of Child Development and Disorders
ChongqingChina

Key Laboratory of Pediatrics in Chongqing
CSTC2009CA5002ChongqingChina

Chongqing International Science and Technology Cooperation Center for Child Development and Disorders
ChongqingChina

Shuang-Zi Li
Department of Neurology, Children's Hospital Affiliated to Chongqing
University of Medical Sciences
ChongqingChina

Ministry of Education Key Laboratory of Child Development and Disorders
ChongqingChina

Key Laboratory of Pediatrics in Chongqing
CSTC2009CA5002ChongqingChina

Chongqing International Science and Technology Cooperation Center for Child Development and Disorders
ChongqingChina

Yue Hu
Department of Neurology, Children's Hospital Affiliated to Chongqing
University of Medical Sciences
ChongqingChina

Ministry of Education Key Laboratory of Child Development and Disorders
ChongqingChina

Key Laboratory of Pediatrics in Chongqing
CSTC2009CA5002ChongqingChina

Chongqing International Science and Technology Cooperation Center for Child Development and Disorders
ChongqingChina

Sant Pau Hospital
Spain

University of Santiago
Chile

Saint Louis University
USA

The efficacy and Tolerability of the clonidine Transdermal Patch in the Treatment for children with Tic Disorders: a Prospective, Open, single-group, self-controlled study

Citation ; : Song P-P, Jiang L, Li X-j, Hong S-Q, Li S-Z and Hu Y (2017) The Efficacy and Tolerability of the Clonidine Transdermal Patch in the Treatment for Children with Tic Disorders: A Prospective, Open, Single-Group, Self-Controlled Study.

Original research Front. Neurol
832February 201710.3389/fneur.2017.00032Received: 10 October 2016 Accepted: 24 January 2017Frontiers in Neurology | www.frontiersin.org Edited by: Reviewed by: *Correspondence: Specialty section: This article was submitted to Movement Disorders, a section of the journal Frontiers in Neurologyclonidine transdermal patchtic disorderschildrenprediction modelefficacy
Background: To evaluate the efficacy and tolerability of a clonidine transdermal patch in the treatment of children with tic disorders (TD) and to establish a predictive model for patients.Methods: Forty-one patients who met the inclusion criteria entered into 12 weeks of prospective, open, single-group, self-controlled treatment with a clonidine transdermal patch. The Yale Global Tic Severity Scale (YGTSS) was employed before therapy (baseline) and at 4, 8, and 12 weeks after therapy.results: (1) The total effect rates of treatment with a clonidine transdermal patch were 29.27, 53.66, and 63.41% at 4, 8, and 12 weeks, respectively. Compared with the baseline, the differences were significant at three different observation periods.(2) Compared to the level of 25% reduction, there were significant decreases in the score-reducing rate of motor tic and total tic severities at 12 weeks. (3) If the disease course was ≤24 months and the motor tic score was <16 at the baseline, there was an effective rate of 100% for treatment with the clonidine transdermal patch. If the disease course was ≤24 months and the motor tic score was >16, there was an effective rate of 57.1%. If the disease course was >24 months and the clinical classification was chronic TD, there was an effective rate of 62.5%. If the disease course was >24 months and the clinical classification was Tourette's syndrome, 90% of the patients were invalid. (4) The main adverse events were rash, slight dizziness, and headache.conclusion: (1) When patients were pretreated with a D2-dopamine receptor antagonist that was ineffective or not tolerated well, switching to a clonidine transdermal patch treatment was effective and safe. (2) A clonidine transdermal patch could be a first-line medication for mild and moderate TD cases that are characterized by motor tics.

inTrODUcTiOn Tic disorders (TD) commonly occur among youth and are classified into three types: TD, chronic motor or chronic vocal/phonic tics (CTD), and Tourette's syndrome (TS). The prevalence of these types is 10-15, 3-4, and 1%, respectively (1). All TD are more common in boys than in girls (male/female ratio of approximately 4:1), and the clinical course may be transient or chronic. Typically, tic onset occurs in early childhood (i.e., 4-12 years), usually at the age of 7-8 years (2,3). It peaks in severity and prevalence in preadolescence (i.e., 10-12 years) and begins to decline during adolescence. Approximately, one-half to two-thirds of adolescents with TS have a decrease in tic severity by early adulthood (4,5). It is characterized by the presence of sudden, rapid, non-rhythmically repetitive and mainly involuntary body movements (motor tics) and/or vocalizations (phonic tics). TD ranges in location (e.g., face and legs) and complexity (e.g., eye blinks, facial grimacing, jaw, shoulder shrugging, multistep movements, and phrases). Most TS patients also suffer from comorbid conditions such as attention deficit hyperactivity disorder (ADHD), obsessive-compulsive disorder, or depression. At present, the pathogenesis of TD remains unclear. Some scholars have suggested that D2-dopamine receptor dysfunction in the basal ganglia may be the major cause of tics. The principal treatment is pharmacological treatment combined with psychological behavior therapy, and individualization is emphasized (6). Many affected children or adolescents do not require pharmacological treatment because their tics do not impair social and academic functioning. When persistent tics impact the quality of life, social and academic functioning and lifetime activities and psychological behavior therapy fails, pharmacological treatment (e.g., D2-dopamine receptor antagonists, selective monoamine antagonists, alpha-2-adrenoreceptor agonists, and other drugs) should be considered (1,(7)(8)(9)(10). D2-dopamine receptor antagonists, such as haloperidol and tiapride, are often effective but poorly tolerated, so their use is limited (1,2,11).

Recently, dysregulation of the noradrenoreceptor system has been suggested as the underlying pathophysiology. Exacerbation due to stress is a characteristic of tics (12). Clonidine, which acts as an alpha-2-adrenoreceptor agonist, has been used to treat hypertensive patients. Small doses of clonidine can alleviate tics by activating presynaptic autoreceptors in the locus ceruleus and then reducing norepinephrine release and turnover. Since the early 1980s, it has been reported that clonidine has beneficial effects in the treatment of TD (13)(14)(15), especially for patients with ADHD (1,16).

A clonidine transdermal patch is a transdermo-therapeutic system that releases drugs at a constant speed successively for 7 days. Compared with the ordinary preparation, a clonidine transdermal patch has no peak or valley plasma concentration, so it can achieve full efficacy and reduce adverse effects. Changing in weekly intervals only vastly improves compliance in children.

We analyzed the clinical data and follow-up results of 41 patients who were pretreated for more than 2 months with D2-dopamine receptor antagonists using a prospective, open, single-group, self-controlled study. This study was undertaken with multiple objectives: (1) to evaluate the efficacy and tolerability of a clonidine transdermal patch in the treatment of TD and (2) to establish a prediction model for subjects that can provide evidence-based recommendations for appropriately choosing clonidine transdermal patches for treatment.

sUBJecTs anD MeThODs subjects

The inclusive criteria were as follows: (1) between the ages of 6 and 18 years, weight more than 20 kg, normal intelligence and either sex; (2) met the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV; American Psychiatric Association, 1994) for TD (17); (3) received pretreatment for more than 2 months with dopamine receptor antagonists but had a Yale Global Tic Severity Scale (YGTSS) score-reducing rate of ≤25% (18) or was not well tolerated; (4) written informed consent and assent were obtained from patients who were more than 7 years old and from their parents.

Subjects were terminated from participation in the study if they met one or more of the following criteria: (1) in violation of the study protocol during the study period; (2) lost to followup during the study period; (3) unused clonidine transdermal patches for more than 2 weeks; (4) occurrence of any serious adverse events.

This study was approved by the ethics committee of the Children's Hospital of Chongqing Medical University and was conducted in accordance with the latest version of the Declaration of Helsinki.

study Design and Measurements

All subjects had a comprehensive and detailed assessment of their current health status, which included pulse rate, blood pressure, respiratory rate, height, and weight, before treatment and at 4, 8, and 12 weeks after therapy. Every subject completed the examination of routine hematology, routine urinalysis, liver and renal functions, fasting blood glucose, and electrocardiogram before therapy. All TD medications were discontinued before therapy for 1 week, and subjects entered the therapy group after completing the washout period. Subjects were prohibited from using alpha-2-adrenoreceptor blockers, antidepressants or other psychoactive medications, psychological behavior therapy, and transcranial magnetic stimulation during the study period. Subjects who did not want to continue to participate in the study because of a poor therapeutic effect were considered invalid. To prevent bias in the statistical results, we used the last observation carried forward (LOCF) to analyze the data so that we did not exclude these patients from the study.

The tic symptoms of each participant were evaluated by an appointed professional using the YGTSS (19) at baseline (after completing the washout period and before therapy) and at weeks 4, 8, and 12.

The Total Tic Score (TTS) = the motor tic score + the phonic tic scores (range = 0-50).

The Global Severity Score (TGSS) = the motor tic score + the phonic tic score + overall impairment rating (range = 0-100). Severity grading was divided into mild, moderate, and severe, and the TGSSs ranged from 0-25, 25-50, and 50-100, respectively.

Drug and Dosing schedule

Clonidine patches were provided by Shanxi RFL Pharmaceutical Co., Ltd. The drug specifications were as follows: 1.0, 1.5, and 2.0 mg sizes. When the weights of the patients were >20 and ≤40, >40 and ≤60, and >60 kg, the doses of the drug were 1.0, 1.5, and 2.0 mg per week, respectively. The clonidine transdermal patch was placed on the back under the shoulder blades, and the two sides were alternated weekly. The old patch was not removed until the new one had been placed for 1 day. evaluation Using the YGISS score that was measured after completion of the washout period before the therapy as the baseline score, we compared the score-reducing rate of four factors at three separate time points, including baseline and the end of weeks 4, 8, and 12. The contents of the evaluation included the score-reducing rate for motor tics, phonic tics, the total tic score, and the overall impairment rating. The definitions were as follows (1) Score-reducing rate for Motor Tic Score = (Motor Tic Score at baseline − Motor Tic Score at a different period)/Motor Tic Score at baseline × 100%; (2) Score-reducing rate for Phonic Tic Score = (Phonic Tic Score at baseline − Phonic Tic Score at a different period)/Phonic Tic Score at baseline × 100%; (3) Score-reducing rate for Total Tic Score = (Total Tic Score at baseline − Total Tic Score at a different period)/Total Tic Score at baseline × 100%; (4) Score-reducing rate for Overall Impairment Rating = (Overall Impairment Rating at baseline − Overall Impairment Rating at a different period)/Overall Impairment Rating at baseline × 100%.

The definition of clinical efficiency was a YGTSS scorereducing rate of >25% reduction. We took the score-reducing rate of the total tic score as the judgment standard for a curative effect. Clinical recovery was defined as the score-reducing rate of the total tic score >75%; obvious improvement as 50% < score-reducing rate ≤75%; improvement as 25% < scorereducing rate ≤50%; and clinical invalidation as score-reducing rate ≤25%. Clinical efficiency = clinical recovery + obvious improvement + improvement.

Data analyses

Statistical analysis was performed using SPSS22.0 statistical software package. The quantitative data were evaluated by paired t-tests, a one-sample t-test, and analysis of variance. The categorical data were evaluated by a chi-squared test and Fisher's exact probability test. We conducted the efficacy prediction for our data by building a decision tree. The LOCF was used as the end point for a participant. Statistical significance was established at p < 0.05.

resUlTs

A total of 41 subjects were enrolled (34 males, 7 females; male/ female ratio 4.8:1; age range 6-16 years, average 9.21 ± 2.07 years, median 9 years).

Clinical classifications were TD in 7 patients (7/41, 17.0%), CTD in 17 patients (17/41, 41.5%), and TS in 17 patients (17/41, 41.5%).

The disease severity grading was moderate in 28 patients (28/41, 68.3%) and severe in 13 patients (13/41, 31.7%).

The course of the disease ranged from 2 to 109 months; the average was 27.36 ± 21.70 months, and the median was 24 months. All subjects had a history of using D2-dopamine receptor antagonists (e.g., tiapride, haloperidol + artane, and tiapride + topamax), the course of which ranged from 2 months to 5+ years. Of the 41 patients, 2 dropped out of the treatment at 4 weeks, and 7 dropped out at 8 weeks due to inefficacy. One patient dropped out at 8 weeks for clinical control of tics.   all Factors of YgTss score-reducing rates at Different Periods

curative effect analysis

The definition of clinic efficiency was a YGTSS score-reducing rate for each factor that was >25% reduction. Compared with the level of 25% reduction, we conducted a one-sample t-test (p < 0.05) and found a significant difference in the score-reducing rate of motor tics and total tics at 12 weeks, whereas there was no significant difference in the score-reducing rate of phonic tics and the overall impairment rating at different stages ( Table 2). Compared with 4 weeks, there was a significant difference in the score-reducing rate for motor tics and total tics at 8 weeks and in each factor at 12 weeks (p < 0.05). Compared with 8 weeks, there was no significant difference in the score-reducing rate for each factor at 12 weeks (p > 0.05). For intraobserver comparisons, there was some improvement in phonic tics and overall impairment ratings at 12 weeks, but these scores did not reach the criteria of clinical efficacy (25% reduction). It is suggested that a clonidine transdermal patch could improve motor tics but has poor results for phonic tics and overall impairment ratings.

Forecast analysis

We defined treatment outcomes depending on whether they were effective (YGTSS score-reducing rate for each factor for >25% reduction) as a dichotomous dependent variable. There were 11 independent variables (e.g., gender, onset age, age at first treatment, the age and weight at enrollment, the duration of the illness, clinical classification, comorbid ADHD, motor tic score at baseline, phonic tic score at baseline, and overall impairment rating at baseline). On that basis, we designed a four-armed decision tree to predict the conditions for effective treatment with a clonidine transdermal patch. The model used a classification and regression tree as the growing method and used the method of cross-validation to avoid excessive fitting. The rightness rate of the model was 82.9%, which indicates high predictive accuracy and reliability. The gain is shown in Figure 1.

The model showed the following: (1) there was a treatment efficacy of 100% with the clonidine transdermal patch if the disease course was ≤24 months and the motor tic score was <16;

(2) there was an efficacy of only 57.1% if the disease course was ≤24 months and the motor tic score was >16; (3) there was an efficacy of 62.5% if the disease course was >24 months and the clinical classification was CTD; and (4) 90% of patients who were treated with a clonidine transdermal patch were invalid if the disease course was >24 months and the clinical classification was TS. The importance of the independent variables in relation to treatment is summarized in Figure 2. The most important variables were classification (100%), course of the disease (78.1%), and motor tics (40.4%).

safety and Tolerability evaluation

During the 12-week test period, adverse events occurred in 4.76% (2/41) of the subjects, but no subject withdrew from the study due to adverse events. One child reported a rash where the clonidine transdermal patch was placed, and another had a hematuria at week 9. The hematuria may have been unrelated to the clonidine transdermal patch because it disappeared before the drug was withdrawn.

As of June 2015, we had successfully followed up with 37 patients. A total of 56.7% (21/37) of the patients willingly chose There were eight cases that had adverse events during followup after the end of the clinical trial. The most common adverse events were rash (33.3%, 7/21), slight dizziness, and headache (9.5%, 2/21, one combined with rash), which were mild in severity and reasonably well tolerated. There were no severe adverse events with regard to changes in systolic or diastolic blood pressure or pulse.

DiscUssiOn

The YGTSS is the most widely used measure for treatment responses for tics. The YGTSS is based on the severity of motor and phonic tics in five separate dimensions: number, frequency, intensity, complexity, and interference. Each of these dimensions is scored on a 0-5 scale according to tic severity. The total tic score (range 0-50), which consists of the symptoms of tics, is derived by adding the total motor tic Score (range 0-25) and the total phonic tic score (range 0-25). The YGTSS also includes a separate impairment rating focused on distress and impairment experienced in interpersonal, academic, and occupational realms, the summation of which is the so-called overall impairment rating (range 0-50). A global severity score (range 0-100) is derived by summing the total motor tic score, the total phonic tic score, and the overall impairment rating. Because the overall impairment rating often remained unchanged over one week, the total tic score is more suitable for assessment of tic symptom severity. Jeon et al. (18) verified that a 25% reduction in the total tic score provided optimal sensitivity (87%) and specificity (84%) for predicting positive responses. Thus, we defined clinical efficacy as a YGTSS scorereducing rate of >25% reduction. We took the score-reducing rate of the total tic score as a judgment standard for a curative effect.

A dysregulation of neurotransmission and abnormal receptors is believed to be the underlying pathophysiology of tics. It is widely acknowledged that dopamine receptor hypersensitivity in the basal ganglia is the pathogenesis of TD. The dopamine D2 receptor antagonist has achieved the most reliable and fastest treatment response compared to other medications and is therefore the mainstay of any pharmacological treatment. Haloperidol, which is a typical dopamine antagonist and is most commonly used, may improve tic symptoms in 70-80% of patients (8,20) and has an A level of evidence (21). However, this agent may be associated with more and more serious side effects than other neuroleptics. The significant adverse effects, especially extrapyramidal symptoms such as acute dystonia or akathisia, sedation, weight gain, and decline in cognitive functions, have led to its discontinuation. Tiapride, although associated with low side effects, is less effective than haloperidol and has a B level of evidence (21). Many studies have suggested that alpha-2 agonists, such as clonidine, are less effective than antipsychotics or atypical neuroleptics, such as haloperidol, aripiprazole, and risperidone (10,22,23). We found that the effective rates of treatment with a clonidine transdermal patch were 29.27, 53.66, and 63.41% at weeks 4, 8, and 12, respectively. Compared with the baseline, significant changes were observed at three different observation periods. Because our subjects were unaffected by or not tolerant to dopamine receptor antagonists, it can be speculated that switching to a clonidine transdermal patch may be effective if dopamine antagonists are ineffective or intolerable. The study of Du et al. (24) showed that clonidine improved tic symptoms in 68.31% of patients after 4 weeks of treatment and showed a significant change compared with placebo. Zhong et al. (25) reported that there were no significant differences in efficacy between clonidine and tiapride. In our studies, compared with week 4 of treatment with a clonidine transdermal patch, significant changes in the effective rate were observed at weeks 8 (p < 0.05) and 12 (p < 0.01). However, there were no significant differences between weeks 8 and 12 (p > 0.05). It was concluded that the onset time of clonidine may be after the first 4 weeks, and the peak time may be at 8-12 weeks. It has been suggested that clonidine needs to be taken for a longer period (3 weeks or more) than haloperidol to lead to improvement (26,27). Thus, withdrawal should not be undertaken because of ineffectiveness within a short time. It has been reported that medications for treatment of TD are helpful initially and have a noticeable effect but leave tics unchanged or worsened after approximately a year or 6 months (28). Our long-term follow-up of 8-17 months showed that 38.1% (8/21) of patients who were treated with a clonidine transdermal patch achieved complete remission of tics and were relapse free. Thus, clonidine had definite efficacy in the long term.

Further analysis of all factors of YGTSS score-reducing rates at different periods showed that compared with the level of 25% reduction, there were no significant differences in the score-reducing rate of four factors, including motor tics, phonic tics, total tics, and overall impairment, at 4 and 8 weeks. Compared with the level of 25%, there were significant differences in the score-reducing rate of motor tics and total tics at 12 weeks. This finding suggested that a clonidine transdermal patch could improve motor tics but had poor efficacy for phonic tics and overall impairment ratings. This finding is similar to the results of studies by Leckman et al. (29,30) and Du et al. (24). Clonidine is more effective for motor tics than for phonic tics, which may be because motor tics are less influenced by psychological factors.

The conditional fluctuation of tics was frequent, and individual differences were remarkable. Treatment protocols should be individualized and analyzed by synthesis according to treatment history, course of the disease, classification, comorbidities, and age (16). Our model showed that the first three important independent variables affecting treatment were classification (100%), course of the disease (78.1%), and motor tics (40.4%). Hanna (31) suggested that dopamine antagonists should be used in treating severe cases and (or) when other pharmaceutical treatments failed to improve the tics. Clonidine can be used as the first-line treatment for mild to moderate cases, especially for children and patients receiving initial treatment. Other studies (32)(33)(34) have suggested that clonidine can also be used as refractory and (or) in severe cases or cases with co-occurring ADHD. Our forecast analysis showed that there was an efficacy of 100% for treatment with a clonidine transdermal patch if the clinical course was ≤24 months and motor tic score was <16. The efficacy was only 57.1% if the clinical course was ≤24 months and the motor tic score was >16. There was an efficacy of 62.5% if the clinical course was >24 months and clinical classification was CTD. However, 90% of patients who were treated with a clonidine transdermal patch were invalid if the clinical course was >24 months and clinical classification was TS. This finding provides the basis for the conditions in which clonidine transdermal patch treatment should be chosen. However, there was a limitation in the predictive value of the model because of the small sample size.

Haloperidol is often poorly tolerated in TD, with only 20-30% of patients continuing to take this medication over time (35) secondary to the serious side effects. Clonidine, however, often has good tolerance (13-15, 22, 24, 26, 30, 36). The most common adverse events with clonidine are sedation and dizziness, which often tend to be mild to moderate in severity (16,26) without evidence of other adverse effects, including cardiac toxicity (16). In our study, the most common adverse events were rash (33.3%, 7/21), slight dizziness and headache (9.5%, 2/21); no patients dropped out of treatment due to adverse effects. In our long-term follow-up of 8-17 months, 56.7% (21/37) of patients continued to take this medication after the 12-week test. This result implies that clonidine has good tolerance and a high level of safety over time.

Our study was an open-label trial that was not placebo controlled. It could be argued that the findings were the result of a placebo effect rather than reflecting the intrinsic activity of the study drugs. However, compared with other diseases with a psychological origin, TD intrinsically has a lower placebo effect (37,38). The duration of illness for the enrolled children ranged from 2 months to several years or longer, and there were no good responses to D2-dopamine receptor antagonists. Furthermore, placebo effects often do not last long. In our study, clonidine continued to improve the symptoms of TD for the 4-12 weeks of the study period and maintained its effects in the long-term follow-up.

In conclusion, when patients were pretreated with a D2-dopamine receptor antagonist that was ineffective or not tolerated well, switching to a clonidine transdermal patch treatment was effective and safe. A clonidine transdermal patch could be a first-line medication for mild and moderate TD cases that are characterized by motor tics.

eThics sTaTeMenT

We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. aUThOr cOnTriBUTiOns P-PS conducted the acquisition and interpretation of the data and was involved in drafting the manuscript; YH conceived the study, participated in its design, and helped to draft the manuscript; LJ critically revised the study for important intellectual content; X-jL, S-QH, and S-ZL participated in the design of the study and performed the statistical analysis. All authors declare that they have no competing interests. All authors read and approved the final manuscript.

FigUre 1 |
1growing method of classification and regression tree.

FigUre 2 |
2The importance of independent variables affecting treatment. transdermal patch after the 12-week test ended, the courses of which lasted between 4 and 17 months.A total of 38.1% (8/21) of patients who were treated with a clonidine transdermal patch obtained complete remission of tics and were relapse free. Although 4.8% (1/21) of the patients achieved complete remission of tics, they recrudesced after 6 months. Our follow-up results showed long-term tolerability for patients with a clonidine transdermal patch.

Clonidine Transdermal Patch for Tic DisordersFrontiers in Neurology | www.frontiersin.org
February 2017 | Volume 8 | Article 32

TaBle 1 |
1changes in efficacy for the clonidine transdermal patch at different treatment stages.Different stages

At 4 weeks, clinical recovery occurred in 1 patient (1/41, 2.44%), obvious improvement occurred in 1 patient (1/41, 2.44%), improvement occurred in 10 patients (10/41, 24.39%), and invalidation occurred in 29 patients (29/41, 70.73%) (Table 1). The total efficacy rate was 29.27% (12/41). A significant difference (p < 0.001) was observed compared with baseline. At 8 weeks, clinical recovery occurred in 3 patients (3/41, 7.32%), obvious improvement occurred in 14 patients (14/41, 34.15%), improvement occurred in 5 patients (5/41, 12.20%), and invalidation occurred in 19 patients (19/41, 46.34%).The total
efficacy rate was 53.66% (22/41). A significant difference was
observed between baseline (p < 0.001) and 4 weeks (χ 2 = 5.025,
p < 0.05).
At 12 weeks, clinical recovery occurred in 15 patients (15/41,
36.59%), obvious improvement occurred in 6 patients (6/41,
14.63%), improvement occurred in 5 patients (5/41, 12.20%), and

TaBle 2 |
2changes in YgTss score-reducing rate (%) at different treatment stages.YgTss score-reducing rate
Different stages

4 weeks (mean ± sD)
8 weeks (mean ± sD)
12 weeks (mean ± sD)
F
p

Motor tics
16.74 ± 24.75
35.55 ± 34.61 ▲
46.65 ± 36.55** ,▲▲▲
8.717
0.000
Phonic tics
−8.50 ± 51.69
16.68 ± 51.13
24.47 ± 57.76 ▲
2.453
0.094
Total tic
8.52 ± 33.94
32.21 ± 38.19 ▲
45.11 ± 41.29** ,▲▲▲
9.816
0.000
Overall impairment rating
12.19 ± 34.31
25.40 ± 37.82
37.60 ± 44.77 ▲▲
4.308
0.016

Clinical effectiveness for treatment was more than 25% in YGTSS score-reducing rate; compared with the lever of 25%, derived from one-sample t-test, **less than 0.01; compared
with 4 weeks, ▲ p < 0.05, ▲▲ p < 0.01, ▲▲▲ p < 0.001; compared with 8 weeks.
YGTSS, the Yale Global Tic Severity Scale.

4

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CELLULAR NEUROSCIENCE PERSPECTIVE ARTICLE BDNF and the maturation of posttranscriptional regulatory networks in human SH-SY5Y neuroblast differentiation
published: 15 October 2014

Belinda J Goldie
School of Biomedical Sciences and Pharmacy
The Centre for Translational Neuroscience and Mental Health
University of Newcastle
CallaghanNSWAustralia

Schizophrenia Research Institute
SydneyNSWAustralia

by: Takumi Takizawa
Carleton University
Canada Reviewed

Gunma University
Japan

School of Biomedical Sciences and Pharmacy
State University of New York
University Drive, Callaghan2308NSWUSA, Australia

Michelle M Barnett
School of Biomedical Sciences and Pharmacy
The Centre for Translational Neuroscience and Mental Health
University of Newcastle
CallaghanNSWAustralia

Murray J Cairns firstname.lastname@example.com
School of Biomedical Sciences and Pharmacy
The Centre for Translational Neuroscience and Mental Health
University of Newcastle
CallaghanNSWAustralia

Schizophrenia Research Institute
SydneyNSWAustralia

by: Takumi Takizawa
Carleton University
Canada Reviewed

Gunma University
Japan

School of Biomedical Sciences and Pharmacy
State University of New York
University Drive, Callaghan2308NSWUSA, Australia

Shawn Hayley
Hariharasubramanian Ramakrishnan
Murray J Cairns
CELLULAR NEUROSCIENCE PERSPECTIVE ARTICLE BDNF and the maturation of posttranscriptional regulatory networks in human SH-SY5Y neuroblast differentiation
published: 15 October 201410.3389/fncel.2014.00325Edited by: *Correspondence:neuronal differentiationMicroRNAsgene expression profilingcell culture techniquesSH-SY5Y cells
The SH-SY5Y culture system is a convenient neuronal model with the potential to elaborate human/primate-specific transcription networks and pathways related to human cognitive disorders. While this system allows for the exploration of specialized features in the human genome, there is still significant debate about how this model should be implemented, and its appropriateness for answering complex functional questions related to human neural architecture. In view of these questions we sought to characterize the posttranscriptional regulatory structure of the two-stage ATRA differentiation, BDNF maturation protocol proposed byEncinas et al. (2000)using integrative whole-genome gene and microRNA (miRNA) expression analysis. We report that ATRA-BDNF induced significant increases in expression of key synaptic genes, brain-specific miRNA and miRNA biogenesis machinery, and in AChE activity, compared with ATRA alone. Functional annotation clustering associated BDNF more significantly with neuronal terms, and with synaptic terms not found in ATRA-only clusters. While our results support use of SH-SY5Y as a neuronal model, we advocate considered selection of the differentiation agent/s relative to the system being modeled. Citation: Goldie BJ, Barnett MM and Cairns MJ (2014)  BDNF and the maturation of posttranscriptional regulatory networks in human SH-SY5Y neuroblast differentiation. Front. Cell. Neurosci. 8:325.

INTRODUCTION

The nervous system, and in particular the brain, arguably represents the most complex area of human biology. Many different cell types are present, interacting in multiplexed combinations, and every circuit is uniquely wired by individual patterns of experience. This complexity continues down to the subcellular level with intricate transportation systems and biomolecular partitioning. Temporo-spatial specificity of mRNA translation in particular is critical for localized protein synthesis as it supports the synaptic remodeling required for synaptic plasticity.

Recent studies have suggested that small, non-coding RNA species known as microRNA (miRNA) play a role in supporting temporo-spatial traffic of neuronal mRNA (Goldie and Cairns, 2012). Many miRNA demonstrate brain-specific (Smirnova et al., 2005;Krichevsky et al., 2006) and region-specific (Hollins et al., 2014) expression and have been shown to regulate key aspects of brain development and neuronal morphology (Giraldez et al., 2005), including patterning and arealisation, dendritic branching (Xu et al., 2008) and spine volume (Schratt et al., 2006). The presence of miRNA in exosomes suggests they may also play a role in activity-driven communication (Fauré et al., 2006).

A key limitation in studies of the nervous system is that ethical considerations and availability of tissue generally restrict these investigations to animal models, relying on in-vitro or ex-vivo preparations, typically of mouse or rat, to extrapolate human function. Although the composition of the post-synaptic density is well conserved among mammals (Bayés et al., 2010), the dynamic component of plasticity provided by mRNA and miRNA is quite divergent. Human transcription networks have evolved a complexity that drives species-specific gene expression in the pre-frontal (PFC) and frontal cortices (Konopka et al., 2012). Among transcripts that are subject to human-specific developmental remodeling, miRNA in the PFC exhibit the greatest and most rapid divergence from other primates with an average evolutionary length 24 times that of other transcript types (Somel et al., 2011). These findings suggest that animal models, while informative, may not provide full understanding of the molecular mechanisms underlying neuronal function in higher primates and humans.

NEURONAL DIFFERENTIATION OF NEUROBLASTOMAS

To overcome this limitation, several human immortalized cell lines are available which for some purposes can model human neuron behavior. Among these, neuroblastoma cell lines are highly accessible and can be matured into terminally differentiated, neuron-like cells using agents such as retinoic acid (RA), enabling clearer determination of molecular and morphological changes induced by differentiation. Treatment with RA is associated with inhibition of proliferation, extension of processes commonly termed neurites (Stio et al., 2001), increased acetylcholinesterase (AChE) activity (Sidell et al., 1984), and enhanced production of synaptic vesicles (Sarkanen et al., 2007); features consistent with neuronal maturation.

In the SH-SY5Y cell line, a sympathetic line containing noradrenaline and neuropeptide Y in dense-core vesicles (Goodall et al., 1997;Ou et al., 1998), three stereoisomers of RA, all-trans (ATRA), 9-cis and 13-cis, are all observed to induce this phenotype, however differential activity at the receptor level appears to impact the differentiation process. ATRA acts at the native retinoic acid receptor (RAR), which binds to retinoic acid response elements (RAREs) in the DNA to alter transcription of RA-activated genes, while the 9-cis isomer acts in a similar manner on the retinoid X receptor (RXR). This difference could explain the observation that although 9-cis RA induces stronger morphological and transcriptional responses these changes are reversed after washout, while the response to ATRA is permanent (Redfern et al., 1994). This finding suggests that ATRA may be a more suitable agent for studying the effects of commitment to a neuronal lineage.

NEURONAL MATURATION WITH BDNF

In the developing brain, the final connectivity of a neuron is determined largely by signals received from the neurotrophin family of proteins, which are expressed in a laminar-specific pattern and influence the growth and complexity of the dendritic tree (McAllister et al., 1995). Of this family, brain-derived neurotrophic factor (BDNF) has a profound effect on pyramidal neurons in cortical layers 4 and 5, in addition to which it has demonstrated the capacity to target mRNAs to the synapse (Tongiorgi et al., 1997;Righi et al., 2000) and stimulate their local translation (Miyata et al., 2005). In particular, polysome profiling of active translation conducted by Schratt and colleagues demonstrated that BDNF is required to stimulate local translation of key synaptic components such as CamKII, NMDA receptors NR1, and NR3, PSD93 and LIMK-1 (Schratt et al., 2004). These findings suggest the employment of BDNF for in vitro neuronal maturation may produce cells more closely matching the phenotype and, importantly, gene expression profile of neurons in vivo.

BDNF is active at the trkB receptor (gene name NTRK2), which is absent from naïve SH-SY5Y cells; its expression can be induced by differentiation with ATRA (Kaplan et al., 1993), and some studies have employed a 5-6 day protocol of concurrent ATRA and BDNF treatments. However, in an arguably seminal paper published in 2000, Encinas and colleagues demonstrated that trkB does not reach peak expression in this cell line until 5 days' exposure to ATRA (Encinas et al., 2000); thus these shortduration, dual-agent protocols may bear limited similarity to mature neurons: a model of sequential treatment with ATRA for 5 days followed by BDNF, that yields neurotrophin-dependent mature cells, may be more physiological.

RATIONALE AND METHODS

We performed a comprehensive review of the literature to understand the use of differentiation protocols for the maturation of SH-SY5Y cells. Specifically, for in vitro research work attempting to emulate mature neuronal behavior, we wanted to know to what extent the sequential treatment with ATRA followed by BDNF was being utilized compared with other differentiation protocols or undifferentiated cells. A search of the PubMed literature for "SH-SY5Y" identified 3419 papers published in English between the publication of the Encinas protocol (2000) and the time of writing (April 2014), from which 410 papers studying the neuroblastoma disease itself were excluded. This list was narrowed to 2307 papers by searching for words associated with differentiation, neurodegeneration and mental health conditions (see Supplementary Figure S1 for full description of review inclusion/exclusion criteria). Surprisingly, 1914 of 2307 papers (83%) did not utilize a differentiation protocol at all, nor was an explanation for using undifferentiated cells given.

Among 393 studies employing a differentiation protocol, the most common differentiation agent was ATRA only (283 of 393 papers, 72%, or 12% of all studies); approximately 16% (65 papers, 3% of all studies) utilized other differentiating agents such as the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA), which has been shown to generate an adrenergic phenotype (Påhlman et al., 1983). Moreover, a remarkably small proportion (45 papers, 11 or 2% of all studies) utilized Encinas' sequential protocol of ATRA differentiation followed by BDNF, despite its compelling evidence: The switch from ATRA to BDNF on day 5 coincides with peak expression of its receptor trkB, however the biochemical impact of this transition has not previously been reported, nor has the gene expression profile been extensively characterized. Moreover, the importance of post-transcriptional regulation of gene expression by miRNA in the control of neuronal differentiation, development, connectivity, and synaptic function is now well established, as reviewed in Goldie and Cairns (2012); a thorough investigation of this two-stage neuronal model in this light is therefore timely.

We undertook comparative genome-wide gene and miRNA expression analyses of naïve, 5-day ATRA-treated and 5-day ATRA-+ 7-day BDNF-treated SH-SY5Y using Affymetrix Exon v1.0 and miRNA v2.0 microarrays. Data were analyzed with Genespring Gx 12 software (Agilent) to identify differentially expressed transcripts with Benjamini-Hochberg correction for multiple testing and a corrected p-value cut-off of 0.05. Microarray results were confirmed by examination of a selection of significantly altered transcripts by qPCR with randomprimed cDNA for gene expression and specific mature miRNA primers for miRNA expression as described previously (Santarelli et al., 2011). Functional analysis of gene expression changes was conducted using the Functional Annotation Clustering (FAC) tool of the DAVID bioinformatics suite (Huang et al., 2008) on lists of genes having significantly different expression (as defined above) at each timepoint. Integrated analysis of altered miRNA expression was conducted using QIAGEN's Ingenuity Pathways Analysis software (IPA, QIAGEN Redwood City, www. qiagen.com/ingenuity). A target analysis was performed on significantly up-and down-regulated miRNA, and the list of targets refined by expression pairing with the list of altered genes. Core analysis was carried out on negatively correlated pairings to investigate significantly altered pathways and functional networks.

To report on biochemical neuronal maturity, we measured the acetylcholinesterase (AChE) activity of samples collected at various timepoints during differentiation and maturation using the Amplex Red AChE assay kit according to the manufacturer's instructions (Invitrogen).

SEQUENTIAL DIFFERENTIATION WITH ATRA THEN BDNF YIELDS A MORE NEURONAL CELL POPULATION MORPHOLOGY

Neuroblast cultures were imaged at each stage of treatment at 10X magnification with an Axiovert inverted microscope (Zeiss) (Figure 1). In line with the results of Encinas and colleagues, cells visually appeared more neural across the two-stage differentiation process. Compared with naïve cells (Figure 1A), cells treated with ATRA for 5 days appeared to have reduced proliferation, took on a more polar morphology and began to extend longer, more robust neurites (Figure 1B). During 7 days subsequent BDNF exposure, cell bodies began to migrate into clusters, some a large as ∼110 μm diameter, while neurites increased in number, size and complexity (Figure 1C).

GENE EXPRESSION

Differentiation with ATRA significantly altered the expression of 46 genes; remarkably, 38 of these were up regulated, characterizing neuronal differentiation as the "switching on" of a genetic programme. BDNF maturation had a much bigger effect on gene expression, significantly altering 265 genes (189 up, 76 down) compared with controls and, importantly, 387 genes (123 up, 264 down) compared with ATRA differentiated samples. As shown in Figure 1D, the genes most strongly induced by ATRA were down regulated by BDNF maturation, while a group showing only mild ATRA induction were further strengthened by BDNF. These included many neuronal genes and components of the miRNA biogenesis machinery (Table 1A), a panel of which were validated by qPCR ( Figure 1F). Strikingly, neuropeptide Y (NPY), which has been reported as non-responsive to ATRA (Påhlman et al., 1995), demonstrated 146-fold increase in expression in response to sequential ATRA + BDNF.

Genes "activated" by each of the ATRA differentiation and BDNF maturation processes were functionally analyzed using the DAVID FAC tool. In ATRA-treated samples the only cluster terms to remain significant after p-value correction related to retinoid metabolism. This is perhaps not surprising given the subsequent down regulation of genes induced by ATRA treatment. In contrast, when compared with both control and ATRA-treated cohorts, BDNF-matured samples were strikingly enriched for neuronally relevant clusters including "neuron projection" (ES = 4.83, corrected p = 0.00027) and "synaptic transmission" (ES = 2.33, corrected p = 0.03). Although some of these terms were present in the ATRA results, they were not significant (Table 1B).

miRNA EXPRESSION

In response to ATRA differentiation, 36 miRNA demonstrated significantly different expression. Unlike gene expression there was no clear directional bias with 20 up and 16 down regulated. Also in contrast to the gene expression results, the miRNA most strongly induced by ATRA were further strengthened by BDNF maturation (Figure 1E). BDNF induced differential expression of 70 miRNA compared to undifferentiated cells (42 down, 28 up). Notably, differentiation dramatically increased the expression of miR-132, a key regulator of dendritic growth and arborisation, as well as known brain-specific miRs -210 and -212, the latter of which inhabits a locus with miR-132. Significant increase in expression of miRs -132 and -212 by BDNF maturation was confirmed by qPCR ( Figure 1F). We also observed ATRA-mediated decrease in miR-17 family expression, consistent with previous observations (Beveridge et al., 2009); all members of this family were further down-regulated by BDNF maturation.

FUNCTIONAL INTEGRATION

The importance of BDNF in shaping the regulatory environment of matured cells was investigated using IPA software. Genes showing two-stage activation (173 genes, Figure 1E, red line) were paired with a target analysis of 44 negatively correlated miRNA (10 miRNA with 42 target genes). Core analysis of these molecules showed a strong enhancement of neuronal functionality, with top functional terms "neurotransmission" (p = 1.97e-07, APP, GRIK2, KCNMB4, PCDHB10, PCDHB14, PCDHB5, PSEN1), "neurological disease" (p = 7.41e-07, APP, BCL2, CHGB, DPYSL3, ESRRG, GABRA3, GRIK2, LGR5, NTRK2, PLK2, PSEN1, SCN3A, VCAN), "synaptic transmission of cells" (p = 1.52e-06, APP, GRIK2, PCDHB10, PCDHB14, PCDHB5, PSEN1), and "synaptogenesis" (p = 1.93e-06, APP, PCDHB10, PCDHB14, PCDHB5). Full results are presented in Supplementary Table S1. Visual representation of this analysis revealed that many of these genes encode proteins that reside in the synaptic membrane (Supplementary Figure S2). Moreover, four families of miRNA (miR-18a,b, miR-17,20a,b, miR-130, and miR-1275) were found to be key hubs providing regulatory support to this functionality.

ACETYLCHOLINESTERASE ACTIVITY

Only modest changes in AChE activity were observed during ATRA-induced differentiation, however maturation with BDNF caused sharp increases in activity ( Figure 1G). Strikingly, AChE activity in cells treated with ATRA for 5 days followed by 1 day of BDNF exposure was higher than in those treated for 7 days with ATRA alone, highlighting the importance of the neurotrophin in development of the neuronal phenotype. Moreover, the magnitude of the response increased with ATRA concentration used to induce differentiation (Supplementary Figure S3), possibly due to the expression of more BDNF receptors at higher ATRA concentrations.

DISCUSSION

Human neuroblast cultures, particularly the SH-SY5Y line, are used extensively to model the basic biology of neurons. They may also have application in high-throughput screening assays of neuroactive compounds. The literature is controversial regarding the need to differentiate SH-SY5Y, as exemplified by an exchange between Luchtman and Song (2010) regarding the latter's conclusions with respect to the applicability of SH-SY5Y differentiation in studying neurotoxicity and neuroprotection in Parkinson's disease (Cheung et al., 2009). What is clear from this exchange is the need to carefully consider experimental methodology and applicability of the cell model used within the context of the research question.

Our results presented here suggest that the sequential ATRA differentiation-BDNF maturation program yields a cell  Table 1A) and contained many key neuronal peptides, receptors and miRNA (see Table 1B).

FIGURE 1 | Continued

Symbols delineate differing two-stage expression patterns. (G) A selection of these transcripts examined by qPCR confirmed that maturation with BDNF drove increased expression (red bars) compared with ATRA differentiation alone (black bars). * p < 0.05; * * p < 0.01; * * * p < 0.001. (H) AChE activity was measured after 1, 3, 5, and 7 days' ATRA treatment, or 5 days' ATRA treatment followed by 1, 3 or 5 days' treatment with BDNF. Increases in AChE activity were most strongly elicited by BDNF maturation; notably, 5 day ATRA + 1 day BDNF evoked more response than an extended 7 day ATRA treatment (red box). Treatments were 10 μM ATRA and 50 ng/ml BDNF throughout. population with significantly more characteristics of mature neurons compared with ATRA treatment alone. We observed obvious morphological similarities with neurons, including intricate and complex neurite structure and evidence of migration with cell bodies organizing into clusters. The cells also displayed substantial enhancement of neuron-associated gene expression, in particular induction of NPY, NTRK2, and CHRM2. There was also evidence of increased posttranscriptional regulation, with elevated expression of miRNA biogenesis machinery including, DROSHA, DGCR8, DICER, and XPO5. This capacity was realized through significantly elevated expression of many miRNA, including key neuronal miRs -132 and -212, supporting the critical roles these molecules play in developing and mature neurons. These individual findings were corroborated at the systems level by the abundance of terms related to neuronal operation and synapse formation derived from the integrated functional analysis. Moreover we have recently shown that miRNA expression and distribution in these cells is rapidly altered in response to potassium-induced depolarisation . A substantial component of this depolarisation-associated change in miRNA was found to be mediated by release of miRNA-enriched exosomes. These higher orders of neural differentiation and function are dependent on neurotrophin signaling. The importance of BDNF and the expression of the trk receptors in shaping the neuronal specialization have been demonstrated from development through maturity and plasticity. In particular, during cortical development this neurotrophin exerts profound effects on layer 4 and 5 neurons, where it significantly increases the number and complexity of dendritic branches (McAllister et al., 1995), axonal length (Labelle and Leclerc, 2000), as well as the number of spines (Bamji et al., 2006) and therefore potential synaptic connectivity. BDNF maturation of SH-SY5Y clearly reproduced this effect in terms of neurite morphology; however it is generally considered that these cells do not form synapses. Studies in neurons have shown that the trkB receptor is required for synapse formation, and ablation of trkB resulted in deficits of synapse formation (Luikart et al., 2005). Further, the interaction between BDNF and the trkB receptor plays an important role in neuronal development (Cheng et al., 2011;Dong et al., 2012), and is important for synaptic plasticity (Kang et al., 1996).

Our systematic review revealed only 2% (45 of 2307) of papers used sequentially differentiated cells, and only 35 of these studies allowed sufficient exposure to ATRA (5 days) to allow for maximal expression of trkB. Without the interaction between BDNF and trkB, which is optimally induced by exposing the cells to ATRA for 5 days, synapse formation is unlikely and has not been reported in conventional culture. However, by pre-treating these cells with ATRA, Agholme and colleagues were able to detect Frontiers in Cellular Neuroscience www.frontiersin.org October 2014 | Volume 8 | Article 325 | 5 synaptic structures and vesicular transport in a 3D gel matrix culture. Synapses were found in samples matured in BDNF alone, however were more numerous when matured with a cocktail containing BDNF, neuregulin β1, nerve growth factor and vitamin D 3 (Agholme et al., 2010). Although more work needs to be done to fully characterize these structures, this finding indicates that with more development this culture technique may provide a system for unraveling some of the complexities of the human neuron maturation and connectivity.

CONCLUSIONS

SH-SY5Y is a flexible culturing system that can be differentiated into several mature neuronal phenotypes, depending on the differentiation agent selected. In our investigation of gene expression and the posttranscriptional regulatory environment of these cells, we found that differentiation combined with BDNF maturation is optimal for generating a phenotype approaching mature neurons. By contrast, the changes induced by ATRA alone appeared to produce an intermediate phenotype between immature neuroblasts and mature neurons, and may be a suitable analog for the study of developing neurons. This system provides a robust in vitro alternative to animal models for investigating some aspects of neuronal function with the advantage of the human genetic context for revealing species-specific higher order complexity.

FIGURE 1 |
1Comparison of single-stage (ATRA) and two-stage (ATRA + BDNF) differentiation of SH-SY5Y. Compared with (A) 5 DIV untreated cultures, cells (B) treated with ATRA for 5 days demonstrate reduced proliferation and take on a more polar appearance, with neurites being extended and networks beginning to develop. (C) After further maturation with BDNF, cell bodies migrate into clusters and the neurite networks become increasingly complex. (D) Heat map (k-means, 4 clusters) clustering genes and miRNA significantly differentially expressed (blue = low expression, yellow = high) during sequential differentiation. C = control, R = ATRA, B = BDNF. Four clusters reveal key expression patterns, in particular the strength of BDNF induction (star). These are summarized as trend lines showing average expression of constituent (E) genes and (F) miRNA; the groups of transcripts demonstrating two-stage induction (red lines) clustered significantly for neuronal functional terms (see

Table 1A |
1ANeuronal genes and miRNA demonstrating increasedexpression after BDNF maturation of ATRA-differentiated SH-SY5Y.

Gene symbol Gene name
Fold-change
(BDNF/ATRA)

NPY a,c
Neuropeptide Y
146
RELN a,b,d,e
Reelin
9.01
NTRK2 b,d
Neurotrophic tyrosine kinase, receptor,
type 2

2.0

GRIK3 a,d,f
Glutamate receptor, ionotropic, kainate 3
3.3
GRIN2B a,b,c,d Glutamate receptor, NMDA, subunit 2B
3.28
CHRM1 a,b
Cholinergic receptor, muscarinic 1
3.23
HTR3C a
Serotonin receptor 3C
3.28
HTR5A a,d,e
Serotonin receptor 5A
3.3
DRD2 a,c,f
Dopamine receptor D2
3.3
HCRTR1 d,e
Hypocretin (orexin) receptor 1
3.1
CACNA1C a,b,d,e Calcium channel, voltage-dependent, 1C
subunit

3.3

NRG1 a,b,d
Neuregulin 1
3.25
DICER1
Dicer 1, ribonuclease type III
2.56
DROSHA a
Drosha, ribonuclease type III
2.94
DGCR8 a
DiGeorge syndrome critical region gene 8
3.04
XPO5
Exportin 5
3.48
MIR132
Hsa-miR-132
3.04
MIR212
Hsa-miR-212
1.78

Disease associations:
a schizophrenia.

b Alzheimer.

c Huntington's.

d bipolar/mood disorders.

e autism spectrum disorders

f Parkinson's.

Table 1B |
1BComparison of significance of neuronal terms derived from functional clustering of genes altered by sequential differentiation and maturation of SH-SY5Y.Term
Corrected p-value

ATRA vs.
BDNF vs.
BDNF vs.

CTRL
CTRL
ATRA

Neuron development
0.238
0.037
5.19E-03

Neuron differentiation
0.397
3.05E-03
6.24E-03

Axon
0.557
3.04E-05
0.019

Axonogenesis
0.750
0.046
2.40 E-03

Cell morphogenesis involved in
neuron differentiation

0.763
0.066
2.31 E-03

Neuron projection
0.769
2.52E-05
2.65E-04

Neuron projection development
0.812
0.065
3.94E-03

Synaptic transmission
n/a
1.25E-04
0.031

Synapse
n/a
7.92E-05
0.079

Frontiers in Cellular Neuroscience www.frontiersin.org October 2014 | Volume 8 | Article 325 | 2
Frontiers in Cellular Neuroscience www.frontiersin.org October 2014 | Volume 8 | Article 325 | 6
SUPPLEMENTARY MATERIALThe Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fncel.2014. 00325/abstract
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Conflict of Interest Statement: The authors declare that the research was con. Conflict of Interest Statement: The authors declare that the research was con-