Striatal Syntaixin 1A Plays a Protective Role Against Iminodipropionitrile Induced Tic Disorder Through Interaction with Dopamine Transporter

An important mechanism of Tic disorder (TD) is dysfunction in the dopamine (DA) system. Our pilot observation found the expression of Syntaxin 1A (STX1A), a presynaptic SNARE complex, changed in the striatum of TD animals. The present study aimed to clarify the biological role of striatal STX1A in the pathological state of TD and the specic mechanism of its regulation of the dopaminergic system. The TD rat model was established using iminodipropionitrile (IDPN). Adenovirus was used to modulate the expression of STX1A and dopamine transporter (DAT) in vivo and vitro. Primary culture of striatal dopaminergic neurons was performed for in-vitro observation of the DA reuptake, CO-IP analysis of the interaction between STX1A and DAT. First, using immunouorescence staining, Western blotting, and qPCR, we found that the IDPN induced TD model had reduced striatal STX1A expression. In vitro, the DA content in the supernatant was signicantly lower in the STX1A overexpressed group, and the intracellular DA content was signicantly higher. Overexpression of STX1A in vivo partially counteracts the IDPN-induced TD-like behaviors, including bite time and head shaking time. Meanwhile, in-vivo knockdown of STX1A can aggravates TD-like behaviors. Further, DAT was overexpressed in vivo, and the TD-like behavior was alleviated. Interestingly, overexpression of DAT in the striatum resulted in increased levels of STX1A. In order to clarify the interaction between DAT and STX1A, the CO-IP analysis was conducted based on the protein of puried striatal dopaminergic neurons. Compared to the IgG control, the blots of DAT and STX1A showed signicant binding of each other. Striatal STX1A expression is decreased in TD development, and STX1A plays an anti-TD role possibly through interaction with DAT, which maintains the DA reuptake. The exorbitant DA signal caused by STX1A inhibition drives the pathological stereotyped behavior.


Introduction
Tic disorder (TD) is a neurodevelopmental movement disorder characterized by multiple motor and vocal tics [1]. According to the development process, it can be divided into transient TD, chronic TD and Tourette syndrome (Tourette syndrome, TS), among which TS is the most typical type. TD affects at least 1% of the population worldwide, and the incidence is still rising [2]. TD is associated with attention de cit hyperactivity disorder, obsessive-compulsive disorder, and emotional disorder, which may severely in uence children's social adaptation ability [3][4][5][6]. Moreover, it can lead to lifelong illnesses. Currently, common treatments include psycho-behavioral therapy and drug therapy; and symptomatic treatment was majorly applied to alleviate patients' tic symptoms, but it cannot fundamentally improve the prognosis.
Overall, this chronic neurobehavioral disorder has unclear pathophysiology. One of the important mechanisms is the dysfunction in the dopamine (DA) system, in particular the striatal dopaminergic neurons or striatal dopamine signal system [7][8][9][10][11]. The relationship between dopaminergic neurons and TD has been reported by different teams. Years of research and clinical practice have proved DA receptor antagonists to be effective agents in the treatment of TS or TD (allowing a signi cant tic reduction of about 70%) [12]. Central DA alterations and dysfunction of the dopaminergic system is an important cause of TD, dopamine D2 receptor is noticed to be associated with the occurrence of TD [13][14][15]; dopaminergic neurons in the striatum may be an essential factor that affects the occurrence of TD [16].
Besides, changes in the structure and function of the striatum were observed under the pathological state of TD [17][18][19][20]. Moreover, imaging studies suggested that TS individuals have presynaptic and postsynaptic striatum dopamine neuron dysfunctions [21]; PET technology showed that in TS patients the ventral striatum dopamine release is increased [22]. Collectively, dopaminergic signaling impacts TD-like behaviors and pathological changes, striatal dopaminergic neurons are closely related to the pathogenesis and treatment of TD. But the direct evidence is not su cient, and the detailed upstream mechanisms are still unclear. The presynaptic dopamine transporter (DAT) is an important factor that modulates the dopaminergic signaling, which recaptures released DA, thereby limiting synaptic DA availability, and maintaining dopaminergic tone. Through DAT, released DA is re-uptaken into nerve terminals, and this is a crucial mechanism of steady-state balance for dopaminergic signaling. For TD treatment, DAT is a valuable exploration target.
To unravel mechanism of TD, a variety of animal models have been developed. Establishment of TD model using iminodipropionitrile (IDPN) is a simple, easy-to-use, and widely applied method [23]. In this study, we established a TD rat model through a short-to-medium administration of IDPN. In this model, neuropsychiatric disorder is developed, characterized by stereotyped repetitive involuntary tics, pedaling, biting, head twitching, shaking claws, continuous rotation, etc. [24][25][26].
In addition, our pilot observation found that the expression of Syntaxin 1A (STX1A), a presynaptic SNARE complex, changed in the striatum of the TD model. The present study aimed to clarify the striatal STX1A alteration in the pathological state of TD, its biological effects on the occurrence of TD, and the speci c mechanism of its regulation of the dopaminergic system.

Materials And Methods
Animals and the TD model The SPF-grade SD rats (male, 6 weeks old, weight 200±10 g) were purchased from Changzhou Cavens Experimental Animal Co., Ltd. All experimental animals were adapted to feed for a week before the formal experiment. First, we conducted a pilot experiment to validate the IDPN induced TD model. Rats in the TD group were intraperitoneally injected with iminodipropionitrile (IDPN, 200 mg/kg, Sigma Chemical Co., St. Louis, MO, USA, at 9:00-11:00, for 7 consecutive days). The control group was injected with the same dose of saline (5 ml/kg/day). In our previous investigation, the stereotypic behavior was triggered by IDPN injection (including biting, head twitching, shaking claws, continuous rotation, etc., and the stereotypic behavior score was signi cantly elevated).
For each batch of animals, after the behavioral observation, animals were sacri ced, and brains were sampled. A part of samples was xed in 4% paraformaldehyde; for the other part of samples, the striatum tissue was separated for each animal and homogenized, the proteins and RNAs were extracted and stored at -80℃.
Primary culture of striatal dopaminergic neurons Two SPF-grade SD pregnant mice were purchased from the Changzhou Cavens Laboratory Animal Co., Ltd., and at least 10 pups were obtained for primary culture of dopaminergic neurons. The 3-to-5-day-old SD rats were soaked in 75% ethanol for 30 min. Each rat was transferred to an ultra-clean table; under the aseptic conditions, the brain tissue was taken out, rinsed with pre-cooled PBS, and placed in a petri dish. Under a microscope, the striatal nucleus was exposed. The striatum was picked off and placed in a sterile EP tube, and PBS was added to rinse the tissue. Next, 0.25% pancreatin was added. After a 10-min digestion, the tissue was pipetted and transferred to a new EP tube. An appropriate amount of H-DMEM complete medium (90% H-DMEM medium + 10% FBS + 1% penicillin/streptomycin) was added, the tissue suspension was pipetted and centrifuged (1000 rpm for 5 min). The supernatant was removed, an appropriate amount of Neurobasal complete medium (90% Neurobasal medium + 10% FBS + 1% penicillin/streptomycin) was added and the single-cell suspension was generated by pipetting. All cells then passed through a 70-µm cell sieve. The ltered suspension was added to the cell culture ask, an appropriate amount of Neurobasal complete medium was supplemented. The plate was placed in a culture incubator (37°C, 5% CO 2 ). One day after the cells were extracted, the medium was changed. Then, the medium was refreshed every 3 days. The striatal dopaminergic neurons were identi ed by the tyrosine hydroxylase (TH) staining.

Realtime qPCR
Total RNA was extracted from the striatum region of the brain using TRIZOL (Takara, 9108), and cDNAs were synthesized using a TAKARA PrimeScript™ RT reagent Kit (Takara, RR047A). PCR ampli cation reactions were conducted using a SYBR Green Supermix system (Takara, RR820A) in a 20 µl reaction containing 0.8 µl primers (0.4 µl of the 10 µM Forward primer and 0.4 µl of the 10 µM Reverse primer, respectively) and 2 µl cDNA. The cycling program was as follow: (1) denaturation at 95°C for 1 minute, (2) 40 cycles of (95°C for 5 s + 55°C for 30 s + 72°C for 30 s), (3) melting curve. All measurements were performed in triplicates. The primers pairs (forward and reverse, respectively) used in ampli cation were as follow.

ELISA
For each well, the cell supernatant was collected and centrifuged at 1000×g for 20 min. Then, the supernatant was stored at 2 to 8°C. The above samples were used to detect the concentration of dopamine in the cell culture supernatant. In addition, the cells in each well were gently washed with precooled PBS for 3 times, 200 µl of lysis buffer was added to each well, cells were lysed by pipetting and the lysis solution centrifuged at 10000×g for 5 min. Next, this supernatant was also stored at 2 to 8°C.
The above samples were used to detect the concentration of intracellular dopamine. The DA ELISA Kit (Wuhan Fine Biotech Co., Ltd, Product code EU0392) was used for measurement of the supernatant dopamine content and intracellular dopamine content of the primary dopaminergic neurons. All ELISA operations followed the o cial instructions.

Western Blotting
The cell samples or rat striatum tissues were homogenized in a lysis buffer with protein inhibitor and PMSF (1 mM). The lysates were centrifuged at 1000 rpm for 5 min, and the supernatant was collected.
An amount of 20 µg protein samples were separated using 10% SDS-PAGE gel, followed by being transferred onto PVDF membranes. The blots were rst blocked in TBST-milk and then incubated with the primary antibody overnight at 4°C. Next, the secondary antibodies conjugated with HRP (1: 1000) were used for 2 h of incubation. The expression of β-actin was used as an endogenous reference. The Tanon ECL Kit was used for chemiluminescence, and the Tanon 5200 chemiluminescence imager was used for image capture.

CO-IP
For each protein sample, 500 µg protein was collected, and the STX1A antibody (1:30) or DAT antibody (1:30) was added. The rabbit IgG was used as a negative control. Each tube of mixture was slightly shacked overnight at 4°C. The remaining total protein was directly used in Western Blot as an Input control. Next, 40 µl of protein A/G agarose beads was added, and the mixture was shacked at 4°C for 4 h to couple the antibody to the agarose beads. Then, the suspension was centrifuged at 4°C for 3 min (1000 g), the supernatant was carefully aspirated off, and the precipitate was collected for analysis. Subsequently, 500 µl of RIPA lysate was added to the precipitate, and the system was homogenized. The lysates were centrifuged at 4°C for 3 min (1000 g), the supernatant was collected, and this step was repeated 3 times. The collected proteins were further added 5×SDS loading buffer and boiled for 5 min. Next, routine Western blotting analysis was performed.

Adenovirus
Three pairs of Adenovirus were produced by Shanghai Genechem Co., Ltd., including the STX1Aoverexpression adenovirus and the corresponding negative control virus (namely, STX1A vs NC), STX1A interfering shRNA adenovirus and corresponding scramble RNA control virus (namely, sh-STX1A vs sh-Control), DAT overexpression adenovirus and corresponding negative control virus (namely, DAT vs NC).

Intra-striatal virus injection
Each rat was xed on a stereotaxic instrument, a micro-injector was used to inject the Adenovirus into the bilateral striatum (5 µl each side, the virus amount for each rat was 10^10 units). The injection site was as follow: AP + 1.0 mm, ML ± 2.5 mm, DV -3.8 mm. TD modeling was performed on the 3rd day after injection. After the behavioral observation, animals were sacri ced, and brain tissue samples were collected. Brains were xed in 4% paraformaldehyde, and the reporter gene (GFP) staining was conducted to con rm the accurate injection of the virus.

Immuno uorescence
The rats were transcardially perfused with 0.01M PBS followed by 50 ml of 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brain tissues were quickly separated and post-xed in 4% paraformaldehyde overnight, then the brain was dehydrated in 20% sucrose (0. and donkey anti-mouse IgG conjugated with CY3 (1:200). Next, sections were incubated with DAPI for nucleus staining for 15 min and washed 3 times for 5 min each. Finally, sections were cover slipped, and images were captured under a uorescence microscope.

Stereotyped behavior test
The stereotyped behavior test was conducted at 9:00-11:00. The light condition during the observation period was consistent throughout the experiment. Each rat was placed in the observation box (diameter = 40 cm, height = 30 cm), and the 5-min recording for bite time, head shaking time, and rotation time were conducted by two observers. Finally, the stereotypy score was assessed using the following criterion: No stereotypy or normal activity (score = 0), Discontinuous circling behavior or occasional head twitching (score = 1), Occasionally vertical dyskinetic head and neck movements, occasional sni ng, licking, and biting (score = 2), Continuous circling behavior, increased body raising, increased sni ng, repetitive grooming (such as paw-to-mouth movements) (score = 3), Increased lateral and vertical dyskinetic head and neck movements (score =4).

Statistical analysis
Results were expressed as means ± standard error. For two-group comparison, Student's t-test was used after the normal distribution test. A P value < 0.05 was considered as statistically signi cant.

Results
TD rats have reduced striatal STX1A expression First, the stereotypic behavior induced by IDPN was con rmed in our previous study. The TD model rats had more counts in biting, head twitching, shaking claws, continuous rotation, and the stereotypic behavior score. This validated protocol was also reported by similar studies [23,27,28]. In this study, we rst focused on the striatal STX1A expression of the TD model. The striatal STX1A expression in the protein and mRNA levels (as well as STX1A expression in striatal dopaminergic neurons) were shown in Figure 1. In the immuno uorescence staining of striatal dopaminergic neurons, the TD group showed signi cantly decreased STXA1 positive and STX1A/TH double positive uorescence ( Figure 1A-C, P < 0.01). Consistently, the total STX1A protein (assessed by Western blot, Figure 1D-1E) and STX1A mRNA (assessed by real-time qPCR, Figure 1F) levels were decreased in the TD model rats (P < 0.01 and 0.05, respectively). Additionally, there were no changes in the expression of some genes closely related with STX1A, such as SNAP25, SYN, and gephyrin (data not shown). Together, STX1A expression is reduced in the IDPN induced TD individuals.
Changes of in-vitro dopamine distribution and release under the overexpression of STX1A in primary dopaminergic neurons In vitro, the rat primary dopaminergic neurons were successfully isolated and puri ed. The cell morphology under the microscope is shown in Figure 2A. The cell body was round or polygonal, and the cells had obvious axons and dendrites. The dendrites ranged from thick to thin, with branches. After cell immuno uorescence identi cation, all puri ed cells expressed TH ( Figure 2B). After transfection with the rat-STX1A-overexpression adenovirus (the transfection e ciencies of the two groups were similar, Figure  2C), real-time qPCR analysis con rmed that the expression of STX1A mRNA was increased to about 5 times ( Figure 2D, P < 0.01). Next, we probed the DA levels in the supernatant and the cell lysate. The DA content in the supernatant was signi cantly lower in the STX1A (overexpressed) group than that the negative control (NC) group ( Figure 2E, P < 0.01). Meanwhile, the intracellular DA content (assessed based on the cell lysate samples) was signi cantly higher in the STX1A group ( Figure 2F, P < 0.05). These results suggested that STX1A may promote the reabsorption of dopamine, and that the down-regulation of STX1A in the TD model may lead to impaired DA reuptake, which further causes a high level of extracellular DA that triggers the TD-like behaviors.

Overexpression of STX1A in vivo partially counteracts the IDPN-induced TD-like behaviors
Furthermore, adenovirus was injected into the rat striatum to overexpress rat STX1A in vivo ( Figure 3A), and then the TD model was induced by IDPN 2 days after adenovirus injection. The elevated STX1A expression was veri ed by real-time qPCR ( Figure 3B, P < 0.01), Western blot ( Figure 3C, P < 0.05) and immuno uorescence ( Figure 3D-E, P < 0.01). Behavioral tests showed that the STX1A group had reduced bite time ( Figure 3F, P < 0.05) and head shaking time ( Figure 3G, P < 0.01). There were no signi cant changes in other TD-like behaviors (such as continuous rotation behavior and stereotypy scores). This result is consistent with the above ndings and implies that a su cient STX1A expression has the effect of resisting the development of TD.
In-vivo knockdown of STX1A aggravates TD-like behaviors Conversely, adenovirus was injected into the striatum to knock down STX1A expression in vivo, and then the TD model was induced after two days of recovery. The decreased STX1A expression was veri ed by real-time qPCR ( Figure 4A, P < 0.01), Western blot ( Figure 4B, P < 0.01) and immuno uorescence ( Figure  4C-D, P < 0.01). The sh-STX1A group had signi cantly aggravated TD-like behaviors versus sh-Control, including the signi cant increases in bite time ( Figure 4E), head shaking time ( Figure 4F), continuous rotation behavior ( Figure 4G), and the stereotypy score ( Figure 4H). These results revealed that when the expression level of STX1A is insu cient, the IDPN-induced TD-like behavior can be further aggravated. And it supports the above conclusion that the striatal STX1A may play an anti-TD role.

Overexpression of DAT in vivo alleviates TD-like behavior and increases STX1A expression
Given that overexpression of STX1A can reduce the DA content of the supernatant and increase the intracellular DA content (Figure 2), we hypothesized that STX1A may be associated with DA reuptake and interact with DAT. Similarly, we overexpressed DAT in the striatum using the rat-DAT-overexpression adenovirus. The expression of DAT was veri ed by immuno uorescence (Figure 5A & 5B). Next, we observed the expression of STX1A and TD-like behavior. Interestingly, overexpression of DAT in the striatum resulted in increased levels of STX1A mRNA ( Figure 5C, P < 0.05) and protein ( Figure 5D, P < 0.01). This result suggests that there may indeed be a protein-protein interaction between STX1A and DAT. Moreover, in the behavioral test, the DAT (overexpressed) group exhibited reduced bite time ( Figure  5E, P < 0.05), a slightly (without signi cant difference) decrease in head shaking time ( Figure 5F), decreased continuous rotation time ( Figure 5G, P < 0.05), and highly signi cantly improved stereotypy score ( Figure 5H, P < 0.01). Collectively, DAT can affect the expression of STX1A, and a high expression of DAT has an anti-TD effect.

DAT and STX1A binding analysis
In order to clarify the interaction between DAT and STX1A, we extracted the total protein of puri ed striatal dopaminergic neurons and conducted the CO-IP analysis. Compared to the IgG control, the blots of DAT and STX1A showed signi cant binding to each other ( Figure 6A). This result con rmed that the protein-protein interaction between DAT and STX1A in striatal dopaminergic neurons is a mechanism of the expression-modulation in uence and the effects on DA reuptake.

Discussion
In this study, we used a rat TD model induced by IDPN to observe the role of striatal STX1A in TD-like behaviors. Our main ndings are: (1) TD rats have reduced striatal STX1A expression; (2) overexpression STX1A promotes dopamine reuptake; (3) Overexpression of STX1A in vivo partially counteracts the IDPNinduced TD-like behaviors, while In-vivo knockdown of STX1A aggravates TD-like behaviors; (4) Overexpression of DAT in vivo alleviates TD-like behavior and increases STX1A expression; (5) DAT and STX1A have a protein interaction. From the above results, the pathogenesis of TD and the mechanism of in uence of STX1A on TD-like behavior are proposed as follow (summarized in Figure 6B). Under the condition of IDPN-induced TD, the expression of STX1A in the striatum is impaired, which in turn affects the reuptake of DA by DAT; to be more speci c, combination of STX1A and DAT is crucial in DA reuptake, impaired STX1A expression cause an accumulation of DA in the synaptic cleft which triggers an abnormal DA signal activation in TD development; and the exorbitant DA signal drives the pathological stereotyped behavior. However, this hypothesis urgently needs experimental con rmation.
STX1A encodes a component of the presynaptic SNARE complex, and it is closely associated with presynaptic vesicle release. It participates in serotonin transporter regulatory, glutamate transport and γaminobutyric acid (GABA) transport [29,30]. STX1A might in uence the serotonergic system during neurodevelopment [30]. It is known that ablation of STX1A may cause disruption of 5-HT-ergic transmission and induce abnormal behavior. Moreover, the JNK2/STX1 interaction is involved in presynaptic NMDA-evoked glutamate release [31]. In the parkinsonian animal model induced by amphetamine, the expression of STX1A decreased in the nucleus accumbens (NAc) while increased in the shell region [32]. Similar to Parkinson's disease, the relationship between STX1A and central nervous system diseases is increasingly noticed [30,33]. Moreover, known disease associated with STX1A include Williams Syndrome, autism, Asperger syndrome, children attention-de cit/hyperactivity disorder, and cryptogenic epilepsy [30,[33][34][35][36][37].
To date, the functional relationship between STX1A and dopaminergic neurons is not fully clear, but the interaction of STX1A to DAT has been noticed in other elds. Referential ndings are as follow. The DAT reuptakes dopamine into presynaptic neurons through sodium-dependent calcium channels to regulate the intensity and duration of dopamine signal activation, and STX1A is believed to be involved in this process [38]. DAT/STX1A interaction can regulate the activity of transport channels and dopaminergic synaptic transmission [39]. Another study showed that combination of STX1A and DAT can promote the release of dopamine, and amphetamine can facilitate their combination, which is mediated by CAMKII [40]. Additionally, a study in 2010 used botulinum neurotoxin C to treat rat striatum tissue, it revealed the binding effect of STX1A and DAT and that STX1A can modulate DA by in uencing the re-uptake role of DAT [41]. However, in recent years, limited studies have been published about the relationship between DAT and STX1A. Together, these literatures indicate that STX1A may affect DA reuptake through DAT, and it may regulate the balance of the dopaminergic system. However, to our best knowledge, no studies have observed the change of STX1A in the striatum tissue of TD individuals.
Given the clear function dopamine release and striatal pathways in TS/TD [42][43][44][45], the role of DAT in TS/TD or similar stereotypic behaviors is also a conceivable concern. So far, it has been widely accepted. For example, DAT is involved in methamphetamine-induced behavioral sensitization in tree shrews [46]. DAT KO mice exhibited a highly stereotyped consummatory behavior; increased dopamine in DAT KO mice not only increased perseveration of bouts and individual lick duration, but also increased the behavioral variability in response to the extinction contingency and the rate of extinction [47]. Moreover, DAT inhibition is necessary for cocaine-induced stereotypy in mice [48]. Notably, an evaluation of the serotonin system and perseverative, compulsive, stereotypical, and hyperactive behaviors was found in the DAT-knockout mice [49]. However, above ndings have not shown the role of striatal DAT or DA. Recently, a Chinese herbal prescription was reported to upregulates the striatal DAT and attenuates stereotyped behavior of Tourette syndrome in rats [50]. In addition, another traditional Chinese medicine was noticed to exert the protective and restorative effects against methamphetamine-induced dopaminergic neurotoxicity: it attenuates the stereotyped responses and restores DAT expression to the normal level [51]. Together, it is a reliable belief that DAT inhibition, as well as the resulting DA accumulation, is a key mechanism underlying TD development.
Still, the present study has some limitations. First, due to the time limitation, we have not performed the test about whether direct interference of STX1A (versus the normal control) may cause TD-like behaviors.
Therefore, it is still unclear the necessary role of STX1A in the pathological process of TD. Besides, the speci c binding manner of the two proteins is not clear in this study, and further exploration is still needed.

Conclusion
Striatal STX1A expression is decreased in TD development, and STX1A plays an anti-TD role possibly through interaction with DAT, which maintains the DA reuptake. The exorbitant DA signal caused by STX1A inhibition drives the pathological stereotyped behavior.

Declarations Data Availability
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Author contributions
Xiumei Liu and Xueming Wang conceived and designed the study. Xiumei Liu andXueming Wang performed the experiments, analyzed the data, and wrote the paper, Aihua Cao and Xiaoling Zhang performed the experiments, analyzed the data. All authors declared that they read and approve manuscript nal version.

Funding
This study was funded by National Natural Science Foundation of China (no. 81871076), Shandong Natural Science Foundation (ZR2017LH038).
Compliance with ethical standards

Con ict of interest
All the authors declare that they have no con ict of interest or nancial ties to disclose.     shows the sh-STX1A group has decreased STX1A expression. In the behavioral test, the sh-STX1A group has signi cant increases in (E) bite time, (F) head shaking time, (G) continuous rotation behavior, and (H) the stereotypy score. * P < 0.05, ** P < 0.01. In the behavioral test, the DAT (overexpressed) group exhibits: (E) reduced bite time, (F) a slightly decrease in head shaking time (P > 0.05), (G) decreased continuous rotation time, and (H) highly signi cantly improved stereotypyscore. * P < 0.05, ** P < 0.01. Figure 6