Striatal Synaptic Dysfunction in Dystonia and Levodopa-Induced Dyskinesia.

This review provides an overview of the synaptic dysfunctions of neuronal circuits and underlying neurochemical alterations observed in the hyperkinetic movement disorders, dystonia and dyskinesia. These disorders exhibit similar changes in expression of synaptic plasticity and neuromodulation. This includes alterations in physical attributes of synapses, synaptic protein expression, and neurotransmitter systems, such as glutamate and gamma-aminobutyric acid (GABA), and neuromodulators, such as dopamine, acetylcholine, serotonin, adenosine, and endocannabinoids. A full understanding of the mechanisms and consequences of disruptions in synaptic function and plasticity will lend insight into the development of these disorders and new ways to combat maladaptive changes.

STRAP regulates alternative splicing fidelity during lineage commitment of mouse embryonic stem cells.

Alternative splicing (AS) is involved in cell fate decisions and embryonic development. However, regulation of these processes is poorly understood. Here, we have identified the serine threonine kinase receptor-associated protein (STRAP) as a putative spliceosome-associated factor. Upon Strap deletion, there are numerous AS events observed in mouse embryoid bodies (EBs) undergoing a neuroectoderm-like state. Global mapping of STRAP-RNA binding in mouse embryos by enhanced-CLIP sequencing (eCLIP-seq) reveals that STRAP preferably targets transcripts for nervous system development and regulates AS through preferred binding positions, as demonstrated for two neuronal-specific genes, Nnat and Mark3. We have found that STRAP involves in the assembly of 17S U2 snRNP proteins. Moreover, in Xenopus, loss of Strap leads to impeded lineage differentiation in embryos, delayed neural tube closure, and altered exon skipping. Collectively, our findings reveal a previously unknown function of STRAP in mediating the splicing networks of lineage commitment, alteration of which may be involved in early embryonic lethality in mice.

Diverse Mechanisms Lead to Common Dysfunction of Striatal Cholinergic Interneurons in Distinct Genetic Mouse Models of Dystonia.

Clinical and experimental data indicate striatal cholinergic dysfunction in dystonia, a movement disorder typically resulting in twisted postures via abnormal muscle contraction. Three forms of isolated human dystonia result from mutations in the TOR1A (DYT1), THAP1 (DYT6), and GNAL (DYT25) genes. Experimental models carrying these mutations facilitate identification of possible shared cellular mechanisms. Recently, we reported elevated extracellular striatal acetylcholine by in vivo microdialysis and paradoxical excitation of cholinergic interneurons (ChIs) by dopamine D2 receptor (D2R) agonism using ex vivo slice electrophysiology in Dyt1ΔGAG/+ mice. The paradoxical excitation was caused by overactive muscarinic receptors (mAChRs), leading to a switch in D2R coupling from canonical Gi/o to noncanonical ß-arrestin signaling. We sought to determine whether these mechanisms in Dyt1ΔGAG/+ mice are shared with Thap1C54Y/+ knock-in and Gnal+/- knock-out dystonia models and to determine the impact of sex. We found Thap1C54Y/+ mice of both sexes have elevated extracellular striatal acetylcholine and D2R-induced paradoxical ChI excitation, which was reversed by mAChR inhibition. Elevated extracellular acetylcholine was absent in male and female Gnal+/- mice, but the paradoxical D2R-mediated ChI excitation was retained and only reversed by inhibition of adenosine A2ARs. The Gi/o-preferring D2R agonist failed to increase ChI excitability, suggesting a possible switch in coupling of D2Rs to ß-arrestin, as seen previously in a DYT1 model. These data show that, whereas elevated extracellular acetylcholine levels are not always detected across these genetic models of human dystonia, the D2R-mediated paradoxical excitation of ChIs is shared and is caused by altered function of distinct G-protein-coupled receptors.SIGNIFICANCE STATEMENT Dystonia is a common and often disabling movement disorder. The usual medical treatment of dystonia is pharmacotherapy with nonselective antagonists of muscarinic acetylcholine receptors, which have many undesirable side effects. Development of new therapeutics is a top priority for dystonia research. The current findings, considered in context with our previous investigations, establish a role for cholinergic dysfunction across three mouse models of human genetic dystonia: DYT1, DYT6, and DYT25. The commonality of cholinergic dysfunction in these models arising from diverse molecular etiologies points the way to new approaches for cholinergic modulation that may be broadly applicable in dystonia.

Strength of cholinergic tone dictates the polarity of dopamine D2 receptor modulation of striatal cholinergic interneuron excitability in DYT1 dystonia.

Balance between cholinergic and dopaminergic signaling is central to striatal control of movement and cognition. In dystonia, a common disorder of movement, anticholinergic therapy is often beneficial. This observation suggests there is a pathological increase in cholinergic tone, yet direct confirmation is lacking. In DYT1, an early-onset genetic form of dystonia caused by a mutation in the protein torsinA (TorA), the suspected heightened cholinergic tone is commonly attributed to faulty dopamine D2 receptor (D2R) signaling where D2R agonists cause excitation of striatal cholinergic interneurons (ChIs), rather than the normal inhibition of firing observed in wild-type animals, an effect known as "paradoxical excitation". Here, we provide for the first time direct measurement of elevated striatal extracellular acetylcholine (ACh) in a knock-in mouse model of human DYT1 dystonia (TorA∆E/+ mice), confirming a striatal hypercholinergic state. We hypothesized that this elevated extracellular ACh might cause chronic over-activation of muscarinic acetylcholine receptors (mAChRs) and disrupt normal D2R function due to their shared coupling to Gi/o-proteins. We tested this concept in vitro first using a broad-spectrum mAChR antagonist, and then using a M2/M4 mAChR selective antagonist to specifically target mAChRs expressed by ChIs. Remarkably, we found that mAChR inhibition reverses the D2R-mediated paradoxical excitation of ChIs recorded in slices from TorA∆E/+ mice to a typical inhibitory response. Furthermore, we recapitulated the paradoxical D2R excitation of ChIs in striatal slices from wild-type mice within minutes by simply increasing cholinergic tone through pharmacological inhibition of acetylcholinesterase (AChE) or by prolonged agonist activation of mAChRs. Collectively, these results show that enhanced mAChR tone itself is sufficient to rapidly reverse the polarity of D2R regulation of ChI excitability, correcting the previous notion that the D2R mediated paradoxical ChI excitation causes the hypercholinergic state in dystonia. Further, using a combination of genetic and pharmacological approaches, we found evidence that this switch in D2R polarity results from a change in coupling from the preferred Gi/o pathway to non-canonical ß-arrestin signaling. These results highlight the need to fully understand how the mutation in TorA leads to pathologically heightened extracellular ACh. Furthermore the discovery of this novel ACh-dopamine interaction and the participation of ß-arrestin in regulation of cholinergic interneurons is likely important for other basal ganglia disorders characterized by perturbation of ACh-dopamine balance, including Parkinson and Huntington diseases, l-DOPA-induced dyskinesia and schizophrenia.

Nitric oxide regulates synaptic transmission between spiny projection neurons.

Recurrent axon collaterals are a major means of communication between spiny projection neurons (SPNs) in the striatum and profoundly affect the function of the basal ganglia. However, little is known about the molecular and cellular mechanisms that underlie this communication. We show that intrastriatal nitric oxide (NO) signaling elevates the expression of the vesicular GABA transporter (VGAT) within recurrent collaterals of SPNs. Down-regulation of striatal NO signaling resulted in an attenuation of GABAergic signaling in SPN local collaterals, down-regulation of VGAT expression in local processes of SPNs, and impaired motor behavior. PKG1 and cAMP response element-binding protein are involved in the signal transduction that transcriptionally regulates VGAT by NO. These data suggest that transcriptional control of the vesicular GABA transporter by NO regulates GABA transmission and action selection.

Synaptic long-term potentiation and depression in the rat medial vestibular nuclei depend on neural activation of estrogenic and androgenic signals.

Estrogenic and androgenic steroids can be synthesised in the brain and rapidly modulate synaptic transmission and plasticity through direct interaction with membrane receptors for estrogens (ERs) and androgens (ARs). We used whole cell patch clamp recordings in brainstem slices of male rats to explore the influence of ER and AR activation and local synthesis of 17ß-estradiol (E2) and 5α-dihydrotestosterone (DHT) on the long-term synaptic changes induced in the neurons of the medial vestibular nucleus (MVN). Long-term depression (LTD) and long-term potentiation (LTP) caused by different patterns of high frequency stimulation (HFS) of the primary vestibular afferents were assayed under the blockade of ARs and ERs or in the presence of inhibitors for enzymes synthesizing DHT (5α-reductase) and E2 (P450-aromatase) from testosterone (T). We found that LTD is mediated by interaction of locally produced androgens with ARs and LTP by interaction of locally synthesized E2 with ERs. In fact, the AR block with flutamide prevented LTD while did not affect LTP, and the blockade of ERs with ICI 182,780 abolished LTP without influencing LTD. Moreover, the block of P450-aromatase with letrozole not only prevented the LTP induction, but inverted LTP into LTD. This LTD is likely due to the local activation of androgens, since it was abolished under blockade of ARs. Conversely, LTD was still induced in the presence of finasteride the inhibitor of 5α-reductase demonstrating that T is able to activate ARs and induce LTD even when DHT is not synthesized. This study demonstrates a key and opposite role of sex neurosteroids in the long-term synaptic changes of the MVN with a specific role of T-DHT for LTD and of E2 for LTP. Moreover, it suggests that different stimulation patterns can lead to LTD or LTP by specifically activating the enzymes involved in the synthesis of androgenic or estrogenic neurosteroids.

Opposite long-term synaptic effects of 17ß-estradiol and 5α-dihydrotestosterone and localization of their receptors in the medial vestibular nucleus of rats.

In brainstem slices of male rats, we examined in single neurons of the medial vestibular nucleus (MVN) the effect of exogenous administration of estrogenic (17ß-estradiol, E2) and androgenic (5α-dihydrotestosterone, DHT) steroids on the synaptic response to vestibular afferent stimulation. By whole cell patch clamp recordings we showed that E2 induced synaptic long-term potentiation (LTP) that was cancelled by the subsequent administration of DHT. Conversely, DHT induced synaptic long-term depression (LTD) that was partially reversed by E2. The electrophysiological findings were supported by immunohistochemical analysis showing the presence of estrogen (ER: α and ß) and androgen receptors (AR) in the MVN neurons. We found that a large number of neurons were immunoreactive for ERα, ERß, and AR and most of them co-localized ERß and AR. We also showed the presence of P450-aromatase (ARO) in the MVN neurons, clearly proving that E2 can be locally synthesized in the MVN. On the whole, these results demonstrate a role of estrogenic and androgenic signals in modulating vestibular synaptic plasticity and suggest that the enhancement or depression of vestibular synaptic response may depend on the local conversion of T into E2 or DHT.

Modulatory role of androgenic and estrogenic neurosteroids in determining the direction of synaptic plasticity in the CA1 hippocampal region of male rats.

Estrogenic and androgenic neurosteroids can rapidly modulate synaptic plasticity in the brain through interaction with membrane receptors for estrogens (ERs) and androgens (ARs). We used electrophysiological recordings in slices of young and adolescent male rats to explore the influence of sex neurosteroids on synaptic plasticity in the CA1 hippocampal region, by blocking ARs or ERs during induction of long-term depression (LTD) and depotentiation (DP) by low-frequency stimulation (LFS) and long-term potentiation (LTP) by high-frequency stimulation (HFS). We found that LTD and DP depend on ARs, while LTP on ERs in both age groups. Accordingly, the AR blocker flutamide affected induction of LTD reverting it into LTP, and prevented DP, while having no effect on HFS-dependent LTP. Conversely, ER blockade with ICI 182,780 (ICI) markedly reduced LTP, but did not influence LTD and DP. However, the receptor blockade did not affect the maintenance of either LTD or LTP. Moreover, we found that similar to LTP and LTD induced in control condition, the LTP unveiled by flutamide during LFS and residual LTP induced by HFS under ICI depended on N-methyl-d aspartate receptor (NMDAR) activation. Furthermore, as the synaptic paired-pulse facilitation (PPF) was not affected by either AR or ER blockade, we suggest that sex neurosteroids act primarily at a postsynaptic level. This study demonstrates for the first time the crucial role of estrogenic and androgenic neurosteroids in determining the sign of hippocampal synaptic plasticity in male rat and the activity-dependent recruitment of androgenic and estrogenic pathways leading to LTD and LTP, respectively.

Influence of sex and estrous cycle on synaptic responses of the medial vestibular nuclei in rats: role of circulating 17ß-estradiol.

We investigated the possible influence of sex and estrous cycle on the synaptic responses of neurons in the medial vestibular nucleus (MVN) and their long-term modifications. In brain stem slices of male and female rats during proestrus (PE) and diestrus (DE), we evaluated the field potential evoked in the MVN by vestibular afferent stimulation. Here we find that in PE females the field potential had a lower threshold and higher amplitude than in DE females and in males and also that the stimulus-response curve was shifted to the left. Such difference is related to the level and cyclic fluctuation of circulating 17ß-estradiol (E(2)). This is supported by the exogenous administration of E(2) in DE females and males, with low levels of circulating E(2) that enhanced the field potential amplitude to values close to those of PE females. Sex and estrous cycle also influence the MVN synaptic plasticity. This has been shown by investigating the effect of testosterone (T) on the induction of long-term effects, since T is the precursor for the neural synthesis of E(2) (estrogenic pathway), which is involved in the induction of fast long-term potentiation (LTP), or of 5α-dihydrotestosterone (DHT, androgenic pathway) which mediates slow LTP and long-term depression (LTD). We found that T mostly induced LTD in PE females and no effect in DE females, while it only provoked fast LTP in males. We suggest that high level of circulating E(2) may interfere with the conversion of T, by inhibiting the neural estrogenic pathway and facilitating the androgenic one. On the whole these results demonstrate an influence of circulating E(2) on vestibular synaptic transmission and plasticity that in some cases may contribute to the sex and menstrual cycle dependence of symptoms in human vestibular pathology.

Long-term potentiation in the rat medial vestibular nuclei depends on locally synthesized 17beta-estradiol.

In male rat brainstem slices, we investigated the involvement of locally synthesized 17beta-estradiol (E(2)) in the induction in the medial vestibular nucleus (MVN) of long-term potentiation (LTP) by high-frequency stimulation (HFS) of the primary vestibular afferents. We demonstrated that the blockade of aromatase by letrozole or of E(2) receptors (ERalpha and ERbeta) by ICI 182,780 prevented the HFS-induced LTP of the N1 wave of the evoked field potential (FP) without affecting baseline responses. Only prolonged afferent activation could induce low LTP. In contrast, HFS applied under a combined blockade of GABA(A) receptors and aromatase or ERs was still able to induce LTP, but it was significantly lower and slower. These findings demonstrate that E(2) does not have a tonic influence on the activity of the MVN neurons and provide the first evidence of the crucial role played by local synthesis of E(2) in inducing LTP. We suggest that the synthesis of E(2) occurs after aromatase activation during HFS and facilitates the development of vestibular synaptic plasticity by influencing glutamate and GABA transmission.

Effects of 17beta-estradiol on synaptic plasticity in the rat medial vestibular nuclei.

CONCLUSION: This study shows that 17beta-estradiol (E(2)) can amplify the long-term potentiation (LTP) induced in the vestibular nuclei by high frequency stimulation (HFS), while potentiation induced by E(2) alone, which is unrelated to synaptic high frequency activation, is reversed by HFS. OBJECTIVE: Like HFS, exogenous E(2) induces long-lasting enhancement of synaptic responses to vestibular afferent stimulation in the medial vestibular nuclei (MVN), through NMDA receptor activation. The aim of this study was to verify the possible interaction of E(2) and HFS in inducing LTP. MATERIALS AND METHODS: In rat brainstem slices, we analysed the modifications induced in the field potential evoked in the MVN by: 1) HFS delivered after induction of E(2) effect and 2) E(2) applied after induction of HFS-LTP. RESULTS: HFS reversed the E(2)-induced potentiation in most cases, while E(2) was able to increase the magnitude of potentiation induced by HFS.