Vesicular Acetylcholine Transporter Alters Cholinergic Tone and Synaptic Plasticity in DYT1 Dystonia

ABSTRACT Background Acetylcholine‐mediated transmission plays a central role in the impairment of corticostriatal synaptic activity and plasticity in multiple DYT1 mouse models. However, the nature of such alteration remains unclear. Objective The aim of the present work was to characterize the mechanistic basis of cholinergic dysfunction in DYT1 dystonia to identify potential targets for pharmacological intervention. Methods We utilized electrophysiology recordings, immunohistochemistry, enzymatic activity assays, and Western blotting techniques to analyze in detail the cholinergic machinery in the dorsal striatum of the Tor1a+/− mouse model of DYT1 dystonia. Results We found a significant increase in the vesicular acetylcholine transporter (VAChT) protein level, the protein responsible for loading acetylcholine (ACh) from the cytosol into synaptic vesicles, which indicates an altered cholinergic tone. Accordingly, in Tor1a+/− mice we measured a robust elevation in basal ACh content coupled to a compensatory enhancement of acetylcholinesterase (AChE) enzymatic activity. Moreover, pharmacological activation of dopamine D2 receptors, which is expected to reduce ACh levels, caused an abnormal elevation in its content, as compared to controls. Patch‐clamp recordings revealed a reduced effect of AChE inhibitors on cholinergic interneuron excitability, whereas muscarinic autoreceptor function was preserved. Finally, we tested the hypothesis that blockade of VAChT could restore corticostriatal long‐term synaptic plasticity deficits. Vesamicol, a selective VAChT inhibitor, rescued a normal expression of synaptic plasticity. Conclusions Overall, our findings indicate that VAChT is a key player in the alterations of striatal plasticity and a novel target to normalize cholinergic dysfunction observed in DYT1 dystonia. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Moreover, pharmacological activation of dopamine D2 receptors, which is expected to reduce ACh levels, caused an abnormal elevation in its content, as compared to controls. Patch-clamp recordings revealed a reduced effect of AChE inhibitors on cholinergic interneuron excitability, whereas muscarinic autoreceptor function was preserved. Finally, we tested the hypothesis that blockade of VAChT could restore corticostriatal long-term synaptic plasticity deficits. Vesamicol, a selective VAChT inhibitor, rescued a normal expression of synaptic plasticity. Conclusions: Overall, our findings indicate that VAChT is a key player in the alterations of striatal plasticity and a novel target to normalize cholinergic dysfunction observed in DYT1 dystonia. © 2021 The Authors.

Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society
Dystonia is a common movement disorder characterized by sustained, repetitive muscle contractions, twisting movements, and abnormal postures. 1 Deletion of three base pairs (ΔGAG) in the TOR1A gene results in the loss of a glutamic acid in the C-terminal of the protein torsinA that is associated to early-onset generalized dystonia (DYT1). 2 Currently, the precise function of torsinA remains unclear, although it plays a role in multiple cellular activities, including protein folding, protein quality control of the endoplasmic reticulum, control of secretion, lipid metabolism, trafficking, degradation, and nuclear envelope dynamics. [3][4][5][6][7] Anticholinergic drugs are among the few pharmacological options for medical treatment of DYT1 dystonia, 8,9 although their use is impeded by a number of serious adverse effects. The rationale behind the use of anticholinergic agents relies on the imbalance between striatal dopamine and acetylcholine (ACh), which results in an increased cholinergic tone. [10][11][12] Although cholinergic interneurons (ChIs) represent a minority of striatal neuronal population, they produce an extensive and dense innervation, providing the highest regional level of ACh and cholinergic markers in the brain and playing a central role in the integration of corticostriatal and thalamostriatal inputs with dopaminergic nigrostriatal innervation. 13,14 In normal conditions, striatal cholinergic tone is downregulated either by acetylcholinesterases (AChE), ACh-degrading enzymes, or by the activation of both muscarinic M2/M4 autoreceptors (M2/M4 mAChR) and dopamine 2 receptors (D2R). Indeed, D2R activation normally reduces the frequency of spontaneous firing of ChIs and inhibits the release of ACh. [15][16][17] There is now robust evidence for a fundamental alteration of D2R signaling in the DYT1 mouse striatum consisting in an excitation, rather than inhibition, of the firing frequency of striatal ChIs. 18,19 The loss of the mutual control between striatal dopamine and ACh and the subsequent neurochemical imbalance are responsible for the impairment of bidirectional corticostriatal synaptic plasticity. Indeed, multiple DYT1 dystonia rodent models showed significant abnormalities in corticostriatal long-term synaptic plasticity, exhibiting the loss of both long-term depression (LTD) and synaptic depotentiation, 20-24 as well as an enhanced long-term potentiation. 20,21 Accordingly, these synaptic plasticity deficits could be compensated either by lowering ACh content or by antagonizing M1 muscarinic receptors. 20,21,23,25 Despite such extensive evidence, the nature of ACh dysregulation remains to be clarified. Here, we utilized a multidisciplinary approach to measure the possible changes in cholinergic markers, and their functional impact in the pathophysiology of dystonia, in the Tor1a null (Tor1a +/À ) mouse model. 26 These mice show a decrease in torsinA levels, 19 as well as electrophysiological and molecular alterations common to multiple rodent models of DYT1 dystonia (for a review, see references [27][28][29]. In Tor1a +/À mice, we identified a significant alteration in the expression of the striatal vesicular acetylcholine transporter (VAChT), which loads ACh into the presynaptic vesicles before its release through exocytosis. Blockade of VAChT restored long-term synaptic depression (LTD) alterations, indicating its functional relevance. Altogether, our results indicate that VAChT is a potential target for pharmacological intervention.

Mouse Model
Male Tor1a +/À null mice 26 (9-to 12-week-old, B6; 129-Tor1atm1Wtd/J, 006251 The Jackson Laboratory) and their wild-type littermates were used for the experiments. The experimental procedures were approved by the Institutional Ethical Review Committee and the Italian Ministry of Health (authorization no. 223/2017-PR) in accordance with the European Union and Italian directives (2010/63EU, D.Lgs 26/2014). The mouse strain was bred at Fondazione Santa Lucia Animal Facility. Mice were housed in groups of 4 per cage in a temperature-controlled room with 12-hour light-dark cycle. Water and food were provided ad libitum.

Gene Expression Analysis by Real-Time Quantitative Polymerase Chain Reaction
Total RNA was extracted from dorsal striata using TRIreagent (Sigma-Aldrich, Merck, Milan, Italy) and quantified using an ND-1000 spectrophotometer (NanoDrop Technologies 2000C, Thermo Scientific, Milan, Italy). RNA integrity was confirmed by 2% agarose gel electrophoresis. Then, 1 mg of total RNA was treated with DNAase I (Invitrogen) and reverse transcribed to complementary cDNA (Roche Italia, Milan, Italy). Real-time PCR (polymerase chain reaction) was performed on 25 ng of cDNA using specific primers (assay ID 316836 Gene Symbol Slc18a3). Quantitative PCR reactions were carried out in duplicate using the SYBR Green I Master Mix (Roche) on a Roche Light Cycler LC480 system. The relative VAChT gene expression was analyzed using the 2 (-ddCt) method. 34

Detection of ACh and AChE Activity
For AChE activity assay, mice were killed by cervical dislocation, their brains were removed, and dorsal striata were rapidly dissected at 0 C and stored at -80 C until further analysis. The AChE activity was determined by a colorimetric method, using the AChE assay kit (ab138871, Abcam). The striatal tissue was homogenized in lysis buffer supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich), with a motor-driven pestle for three cycles, followed by alternate freezing and thawing of the samples. Dorsal striatum ACh levels were measured using a choline/ACh assay kit (ab65345, Abcam). Absorbance was measured using a Microplate Reader (Multiskan GO, Thermo Scientific). For the measurement of basal ACh content, slices (200-400 μm thick) of dorsal striatum were immediately lysed; in another set of experiments, slices were incubated for 4 minutes at room temperature with 10 μM quinpirole in oxygenated aCSF.

Drugs
Drugs were bath applied and diluted at the final concentration in aCSF. Vesamicol and neostigmine were obtained from Merck, Milan, Italy. Picrotoxin and donepezil were purchased from Bio-techne, Milan, Italy.

Statistical Analysis
Excitatory postsynaptic current (EPSC), Excitatory postsynapticpotential (EPSP), and action potential amplitudes were measured using Clampfit (pClamp 10, Molecular Devices). Statistical analysis was performed using GraphPad Prism. Data were obtained from at least three independent biological samples. All biological replicates are represented by "N," number of animals, and "n," number of cells. Values in the text and in the figures are presented as mean AE standard error of the mean. Two-tailed paired or unpaired t test was used for two-sample comparisons. F test was used to compare dose-response curves. The significance level was set at P < 0.05*, P < 0.01**, and P < 0.001***.

VAChT Protein Level Is Increased in the Dorsal
Striatum of Tor1a +/À Mice A functional cholinergic neurotransmission is maintained through an appropriate synthesis, vesicular packaging, and release of ACh. Our confocal microscope analysis showed that VAChT is localized to the soma of large-sized ChIs in Tor1a +/À striatum, similar to wild-type mice (not shown) as well as to the known distribution of the ACh synthesizing enzyme ChAT ( Fig. 1A-C). In addition, VAChT immunostaining was abundantly present in axonal varicosities, as punctiform labeling (Fig. 1B,C). Indeed, highmagnification images show coexpression of VAChT and ChAT in ChIs, consistent with previous observations. 35, 36 We then evaluated CHT1, ChAT, and VAChT protein expression levels in the dorsal striatum. As previously reported, torsinA protein level was reduced in Tor1a +/À mice compared to controls ( Fig. 1D; P < 0.0001***). CHT1 (Fig. 1E) and ChAT (Fig. 1F) protein levels were not significantly different in Tor1a +/+ versus Tor1a +/À striatum (CHT1, P = 0.47; ChAT, P = 0.85). Striatal ChIs coexpress VGLUT3. 37,38 VGLUT3 protein level was similar in the striatum of wild-type and mutant mice ( Fig. 1G; VGLUT3, P = 0.93). VAChT immunoreactivity revealed an intense band at the predicted size of 70 kDa (Fig. 1H). Quantification of band intensity revealed a significant increase in VAChT protein level in Tor1a +/À mice, as compared to Tor1a +/+ control mice ( Fig. 1H; P = 0.0176*). Therefore, we verified whether the increase in VAChT protein abundance was caused by an enhancement in mRNA level. However, we found no difference in the expression of VAChT (Slc18a3) mRNA between Tor1a +/À and Tor1a +/+ mice ( Fig. 1I; P = 0.23). Our findings provide evidence for a selective increase in VAChT protein level in the dorsal striatum of Tor1a +/À in spite of no significant changes in mRNA expression.
ACh Content Is Elevated in the Striatum of Tor1a +/À Mice Both in Basal Condition and after D2R Activation These findings prompted us to measure ACh content in striatal slices from Tor1a +/À mice. To minimize ACh degradation, we quickly collected the samples in liquid nitrogen. By means of a colorimetric assay, we measured an increase in striatal ACh content in Tor1a +/À with respect to wild-type mice ( Fig. 2A; P = 0.0293*). We previously found that the activation of D2R increases the frequency of ChI spontaneous firing activity in Tor1a +/À mice, 19 similar to that reported in multiple DYT1 models. 12,18,21,32,39 To verify that D2R-dependent increase in firing frequency would result in an elevation in ACh release in Tor1a +/À mice, we measured ACh content after striatal slice treatment with the D2R agonist quinpirole. Bath application of quinpirole (10 μM, 4 minutes) did not significantly affect ACh content in wild-type slices, whereas it increased striatal ACh level in Tor1a +/À striatum ( Fig. 2B; P = 0.001**). Overall, these data show that basal ACh content is enhanced in the striatum of Tor1a +/À mice, in line with microdialysis experiments performed in Tor1a +/Δgag mice. 12 Furthermore, our experiments demonstrate that D2R activation abnormally elevates ACh tone in Tor1a +/À mice.
To investigate whether the abnormal D2R activity might cause the increase in VAChT protein level observed in Tor1a +/À mice, we treated dorsal striatum slices with the D2R antagonist sulpiride (10 μM, 6 hours, 32 C). This prolonged treatment did not alter VAChT protein level with respect to untreated, contralateral slices (Fig. S1A; Tor1a +/+ P = 0.433; Tor1a +/À P = 0.431). There was no statistically significant difference in the effect of sulpiride on VAChT protein expression between genotypes (P = 0.94). To further rule out the role of the enhanced striatal ACh tone in the increase in VAChT, we performed a treatment of slices with hemicholinium (10 μM, 6 hours, 32 C). Similar to sulpiride, hemicholinium did not alter VAChT protein level with respect to untreated, contralateral slices (Fig. S1B; Tor1a +/+ P = 0.40, Tor1a +/À P = 0.81). There was no statistically significant difference in the effect of the treatment with hemicholinium between genotypes (P = 0.63). AChE Activity Is Increased in Tor1a +/À Mice AChE is the enzyme that catalyzes the rapid breakdown of ACh in the striatum. 40,41 By means of a colorimetric assay, we therefore measured the enzymatic activity and found a significant increase in AChE activity in Tor1a +/À compared to wild-type mice ( Fig. 2C; P = 0.048*). These results indicate that the increased rate of enzymatic degradation is likely a compensatory mechanism triggered by the elevation of basal ACh levels.

Inhibition of VAChT Restores Striatal Long-Term Depression
A prominent involvement of cholinergic transmission in the impairment of striatal synaptic plasticity has been shown in DYT1 dystonia models, where a loss of corticostriatal LTD has been reported. 20,21,[23][24][25] We investigated whether, by blocking VAChT and therefore reducing ACh uptake into synaptic vesicles and, in turn, ACh release, we might rescue corticostriatal LTD. EPSPs were evoked by 0.1-Hz stimulation of corticostriatal fibers. Slice perfusion with vesamicol (20 μM, 20 minutes), a VAChT-blocking agent, did not modify the intrinsic membrane properties of medium spiny neurons (MSNs) (not shown), as well as the amplitude and paired-pulse ratio (PPR) of corticostriatal EPSPs in both genotypes (Fig. 4A,B; Tor1a +/+ P = 0.268; Tor1a +/À P = 0.682).
These data support the hypothesis that an increased presynaptic VAChT protein level elevates the vesicular packaging of ACh, thus allowing an enhanced release in the dorsal striatum and generating an increased ACh tone, which prevents LTD induction (Fig. 5).

Discussion
The paucity of novel drugs for the treatment of dystonia is determined by the lack of definite therapeutic targets. Although dystonia is not typically associated with degeneration or obvious neuropathological features, striatal dysfunction, including abnormal cholinergic transmission, is consistently implicated in multiple forms of dystonia, including DYT1 dystonia. The aim of the present study was to better define the mechanistic basis for such abnormality in the DYT1 striatum. Our results indicate that the striatal cholinergic machinery is altered at multiple levels in Tor1a +/À mice, ultimately producing a major elevation in ACh content.
ACh is synthesized by ChAT from the substrates choline (taken up into the cell by the high-affinity choline transporter CHT1) and acetylCoA. ACh is then packaged into vesicles by VAChT, which is encoded by a small gene embedded in the gene-producing ChAT, and its action is terminated by AChE. Although we found no significant differences in ChAT or CHT1 protein expression, we measured an increase in VAChT protein amounts in mutant mice, without changes in mRNA levels. In fact, protein abundance is regulated at different levels, including protein degradation. 46 The finding of an increased level of the transporter despite the fact that the synthesizing enzyme is unaffected is not surprising. Previous studies found that an increased protein level of VAChT does not imply the involvement of other cholinergic markers. 36,47,48 Given the critical role of VAChT in cholinergic signaling, as a rate-limiting factor for replenishing cholinergic vesicles, our data show that an increased VAChT activity may be able to sustain enhanced ACh storage and release from synaptic vesicles in Tor1a +/À mice. These findings are in agreement with previous observations showing that a similar increase in VAChT protein level leads to an increase in cholinergic tone. 48,49 Accordingly, transgenic VAChT-deficient mice show decreased basal and evoked ACh release in the striatum. 50 Furthermore, VAChT overexpression induces changes in the morphology of striatal ChIs. 36 Interestingly, these observations are consistent with a previous work reporting an enlargement of striatal ChIs in a knock-in mouse model of DYT1 dystonia. 51 Spontaneous firing activity of ChIs ensures a basal cholinergic tone in the striatum, 52-54 which is negatively modulated by M2/M4 muscarinic autoreceptors and D2R dopamine receptors. Basal firing frequency was not altered in Tor1a +/À ChIs, and the muscarinic autoreceptor response was conserved, as previously reported. 12,19,55 AChE inhibition, by increasing endogenous ACh content, caused a decrease in ChI spontaneous firing activity in both wild-type and mutant striatal slices. However, the decrease in firing rate in Tor1a +/À ChIs was less pronounced compared to Tor1a +/+ . This observation is in accordance with present data showing an increased AChE activity in Tor1a +/À striatum and in line with previous reports from a different DYT1 mouse model. 20 We hypothesize that the lack of changes in ChIs basal firing frequency, despite an increased ACh tone, might be due to the overactivity of AChE, preventing an increased activation of muscarinic autoreceptors. Such an increase in AChE activity may represent a compensatory mechanism to limit the abundance of ACh in the synaptic cleft. The physiological relevance of the increased ACh tone was demonstrated by our observation that, on AChE maximal inhibition, the amplitude of GABAergic synaptic currents was decreased more in Tor1a +/À than in wild-type ChIs.
Similar to M2/M4 autoreceptors, in physiological conditions also D2R activation exerts an inhibitory action on striatal ChI firing activity. However, in several DYT1 rodent models we and others reported that the activation of D2R causes a significant increase in ChI firing rate. 12,18,19 Here we demonstrate that the activation of striatal D2R produces a significant increase in ACh content in striatal slices of Tor1a +/À mice. Indeed, a "paradoxical excitation" of ChIs induced by the activation of D2R has been reported also in DYT6 and DYT25 mouse models of genetic dystonia. 56 In the striatum, dopaminergic and cholinergic signaling act synergistically and reciprocally to shape synaptic plasticity. In the Tor1a +/À mouse model, we previously reported the loss of LTD and suggested that this impairment could result from a cholinergic overactivity, because it can be reverted by lowering ACh content or inhibiting postsynaptic muscarinic M1 receptors. 20 In support of this hypothesis, the present data show that VAChT inhibition by vesamicol was able to rescue LTD expression in Tor1a +/À MSNs. Indeed, an increased level of VAChT protein allows either synaptic vesicles to be filled with more ACh or more vesicles to be filled and released, therefore supporting a sustained release under HFS. Because vesamicol did not change corticostriatal PPR, we hypothesize that VAChT inhibition, by limiting ACh release, may dampen the activation of postsynaptic M1 receptors located on MSNs, thus rescuing LTD expression, in accordance with our previous work. 25 Indeed, though the anticholinergic drug trihexyphenidyl, currently used in the management of dystonia, does not display selectivity for M1 receptors, 57 we showed that only M1-selective and M1-preferring antagonists were able to offset synaptic plasticity deficits in DYT1 mice. 25 The mechanisms by which mutant torsinA leads to the specific modification of cholinergic transmission with an increased protein level of VAChT and long-lasting alterations in circuit function are still unknown. The high expression of torsinA in striatal ChIs might explain a preferential vulnerability of these neurons. 11,58 Recent studies suggested a relationship between the loss of ChIs and the expression of a dystonia phenotype in torsinA conditional knockout models. 59,60 However, there is little evidence supporting the degeneration of cholinergic neurons in DYT1 dystonia patients. 59 Histological studies in DYT1 patients' striata indicate a preservation of large aspiny interneurons, which are believed to correspond to ChIs. [61][62][63] Novel, selective VAChT Positron emission tomography (PET) ligands have been recently developed to measure cholinergic function in a number of conditions, including Parkinson's and Alzheimer's diseases. 64,65 VAChT represents a promising target to measure cholinergic deficits in dystonia patients. 66,67 Overall, our findings provide a direct demonstration of a significant alteration of the cholinergic transmission in the striatum of DYT1 mice, further adding to substantial evidence in support of the central role of ACh in DYT1 dystonia pathophysiology.