Rescue of striatal long-term depression by chronic mGlu5 receptor negative allosteric modulation in distinct dystonia models.

An impairment of long-term synaptic plasticity is considered as a peculiar endophenotype of distinct forms of dystonia, a common, disabling movement disorder. Among the few therapeutic options, broad-spectrum antimuscarinic drugs are utilized, aimed at counteracting abnormal striatal acetylcholine-mediated transmission, which plays a crucial role in dystonia pathophysiology. We previously demonstrated a complete loss of long-term synaptic depression (LTD) at corticostriatal synapses in rodent models of two distinct forms of isolated dystonia, resulting from mutations in the TOR1A (DYT1), and GNAL (DYT25) genes. In addition to anticholinergic agents, the aberrant excitability of striatal cholinergic cells can be modulated by group I metabotropic glutamate receptor subtypes (mGlu1 and 5). Here, we tested the efficacy of the negative allosteric modulator (NAM) of metabotropic glutamate 5 (mGlu) receptor, dipraglurant (ADX48621) on striatal LTD. We show that, whereas acute treatment failed to rescue LTD, chronic dipraglurant rescued this form of synaptic plasticity both in DYT1 mice and GNAL rats. Our analysis of the pharmacokinetic profile of dipraglurant revealed a relatively short half-life, which led us to uncover a peculiar time-course of recovery based on the timing from last dipraglurant injection. Indeed, striatal spiny projection neurons (SPNs) recorded within 2 h from last administration showed full expression of synaptic plasticity, whilst the extent of recovery progressively diminished when SPNs were recorded 4-6 h after treatment. Our findings suggest that distinct dystonia genes may share common signaling pathway dysfunction. More importantly, they indicate that dipraglurant might be a potential novel therapeutic agent for this disabling disorder.

Abnormal striatal plasticity in a DYT11/SGCE myoclonus dystonia mouse model is reversed by adenosine A2A receptor inhibition.

Striatal dysfunction is implicated in many movement disorders. However, the precise nature of defects often remains uncharacterized, which hinders therapy development. Here we examined striatal function in a mouse model of the incurable movement disorder, myoclonus dystonia, caused by SGCE mutations. Using RNAseq we found surprisingly normal gene expression, including normal levels of neuronal subclass markers to strongly suggest that striatal microcircuitry is spared by the disease insult. We then functionally characterized Sgce mutant medium spiny projection neurons (MSNs) and cholinergic interneurons (ChIs). This revealed normal intrinsic electrophysiological properties and normal responses to basic excitatory and inhibitory neurotransmission. Nevertheless, high-frequency stimulation in Sgce mutants failed to induce normal long-term depression (LTD) at corticostriatal glutamatergic synapses. We also found that pharmacological manipulation of MSNs by inhibiting adenosine 2A receptors (A2AR) restores LTD, again pointing to structurally intact striatal circuitry. The fact that Sgce loss specifically inhibits LTD implicates this neurophysiological defect in myoclonus dystonia, and emphasizes that neurophysiological changes can occur in the absence of broad striatal dysfunction. Further, the positive effect of A2AR antagonists indicates that this drug class be tested in DYT11/SGCE dystonia.

Exposure to low-dose rotenone precipitates synaptic plasticity alterations in PINK1 heterozygous knockout mice.

Heterozygous mutations in the PINK1 gene are considered a susceptibility factor to develop early-onset Parkinson's disease (PD), as supported by dopamine hypometabolism in asymptomatic mutation carriers and subtle alterations of dopamine-dependent striatal synaptic plasticity in heterozygous PINK1 knockout (PINK1(+/-)) mice. The aim of the present study was to investigate whether exposure to low-dose rotenone of heterozygous PINK1(+/-) mice, compared to their wild-type PINK1(+/+) littermates, could impact on dopamine-dependent striatal synaptic plasticity, in the absence of apparent structural alterations. Mice were exposed to a range of concentrations of rotenone (0.01-1mg/kg). Chronic treatment with concentrations of rotenone up to 0.8mg/kg did not cause manifest neuronal loss or changes in ATP levels both in the striatum or substantia nigra of PINK1(+/-) and PINK1(+/+) mice. Moreover, rotenone (up to 0.8mg/kg) treatment did not induce mislocalization of the mitochondrial membrane protein Tom20 and release of cytochrome c in PINK1(+/-) striata. Accordingly, basic electrophysiological properties of nigral dopaminergic and striatal medium spiny neurons (MSNs) were normal. Despite the lack of gross alterations in neuronal viability in chronically-treated PINK1(+/-), a complete loss of both long-term depression (LTD) and long-term potentiation (LTP) was recorded in MSNs from PINK1(+/-) mice treated with a low rotenone (0.1mg/kg) concentration. Even lower concentrations (0.01mg/kg) blocked LTP induction in heterozygous PINK1(+/-) MSNs compared to PINK1(+/+) mice. Of interest, chronic pretreatment with the antioxidants alpha-tocopherol and Trolox, a water-soluble analog of vitamin E and powerful antioxidant, rescued synaptic plasticity impairment, confirming that, at the doses we utilized, rotenone did not induce irreversible alterations. In this model, chronic exposure to low-doses of rotenone was not sufficient to alter mitochondrial integrity and ATP production, but profoundly impaired the expression of long-term plasticity at corticostriatal synapses in PINK1 heterozygous knockout mice, suggesting that disruption of synaptic plasticity may represent an early feature of a pre-manifesting state of the disease, and a potential tool to test novel neuroprotective agents.

Dopamine-dependent CB1 receptor dysfunction at corticostriatal synapses in homozygous PINK1 knockout mice.

Recessive mutations in the PTEN-induced putative kinase 1 (PINK1) gene cause early-onset Parkinson's disease (PD). We investigated the interaction between endocannabinoid (eCB) and dopaminergic transmission at corticostriatal synapses in PINK1 deficient mice. Whole-cell patch-clamp and conventional recordings of striatal medium spiny neurons (MSNs) were made from slices of PINK1(-/-), heterozygous PINK1(+/-) mice and wild-type littermates (PINK1(+/+)). In PINK1(+/+) mice, CB1 receptor (CB1R) activation reduced spontaneous excitatory postsynaptic currents (sEPSCs). Likewise, CB1R agonists (ACEA, WIN55,212-3 and HU210) induced a dose-dependent reduction of cortically-evoked excitatory postsynaptic potential (eEPSP) amplitude. While CB1R agonists retained their inhibitory effect in heterozygous PINK1(+/-) mice, conversely, in PINK1(-/-) mice they failed to modulate sEPSC amplitude. Similarly, CB1R activation failed to reduce eEPSP amplitude in PINK1(-/-) mice. Parallel biochemical measurements revealed no significant difference in the levels of the two main eCBs, 2-arachidonoylglycerol (2-AG) and anandamide (AEA) in PINK1(-/-) striata. Similarly, no change was observed in the enzymatic activity of both fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), responsible for eCB hydrolysis. Instead, a significant reduction of binding ability of CB1R agonists was found in PINK1(-/-) mice. Notably, the CB1R-dependent inhibition of synaptic activity was restored either by amphetamine or after chronic treatment with the D2 dopamine receptor agonist quinpirole. Additionally, CB1R binding activity returned to control levels after chronic pretreatment with quinpirole. Consistent with the hypothesis of a close interplay with dopaminergic neurotransmission, our findings show a CB1R dysfunction at corticostriatal synapses in PINK1(-/-), but not in PINK1(+/-) mice, and provide a mechanistic link to the distinct plasticity deficits observed in both genotypes.

Negative allosteric modulation of mGlu5 receptor rescues striatal D2 dopamine receptor dysfunction in rodent models of DYT1 dystonia.

Early onset torsion dystonia (DYT1) is an autosomal dominantly inherited disorder caused by deletion in TOR1A gene. Evidence suggests that TOR1A mutation produces dystonia through an aberrant neuronal signalling within the striatum, where D2 dopamine receptors (D2R) produce an abnormal excitatory response in cholinergic interneurons (ChIs) in different models of DYT1 dystonia. The excitability of ChIs may be modulated by group I metabotropic glutamate receptor subtypes (mGlu1 and 5). We performed electrophysiological and calcium imaging recordings from ChIs of both knock-in mice heterozygous for Δ-torsinA (Tor1a(+/Δgag) mice) and transgenic mice overexpressing human torsinA (hMT1). We demonstrate that the novel negative allosteric modulator (NAM) of metabotropic glutamate 5 (mGlu) receptor, dipraglurant (ADX48621) counteracts the abnormal membrane responses and calcium rise induced either by the D2R agonist quinpirole or by caged dopamine (NPEC-Dopamine) in both models. These inhibitory effects were mimicked by two other well-characterized mGlu5 receptor antagonists, SIB1757 and MPEP, but not by mGlu1 antagonism. D2R and mGlu5 post-receptor signalling may converge on PI3K/Akt pathway. Interestingly, we found that the abnormal D2R response was prevented by the selective PI3K inhibitor, LY294002, whereas PLC and PKC inhibitors were both ineffective. Currently, no satisfactory pharmacological treatment is available for DYT1 dystonia patients. Our data show that negative modulation of mGlu5 receptors may counteract abnormal D2R responses, normalizing cholinergic cell excitability, by modulating the PI3K/Akt post-receptor pathway, thereby representing a novel potential treatment of DYT1 dystonia.

Regional specificity of synaptic plasticity deficits in a knock-in mouse model of DYT1 dystonia.

DYT1 dystonia is a movement disorder caused by a deletion in the C-terminal of the protein torsinA. It is unclear how torsinA mutation might disrupt cellular processes encoding motor activity, and whether this impairment occurs in specific brain regions. Here, we report a selective impairment of corticostriatal synaptic plasticity in knock-in mice heterozygous for Δ-torsinA (Tor1a(+/Δgag) mice) as compared to controls (Tor1a(+/+) mice). In striatal spiny neurons from Tor1a(+/Δgag) mice, high-frequency stimulation failed to induce long-term depression (LTD), whereas long-term potentiation (LTP) exhibited increased amplitude. Of interest, blockade of D2 dopamine receptors (D2Rs) increased LTP in Tor1a(+/+) mice to a level comparable to that measured in Tor1a(+/Δgag) mice and normalized the levels of potentiation across mouse groups. A low-frequency stimulation (LFS) protocol was unable to depotentiate corticostriatal synapses in Tor1a(+/Δgag) mice. Muscarinic M1 acetylcholine receptor (mAChR) blockade rescued plasticity deficits. Additionally, we found an abnormal responsiveness of cholinergic interneurons to D2R activation, consisting in an excitatory response rather than the expected inhibition, further confirming an imbalance between dopaminergic and cholinergic signaling in the striatum. Conversely, synaptic activity and plasticity in the CA1 hippocampal region were unaltered in Tor1a(+/Δgag) mice. Importantly, the M1 mAChR-dependent enhancement of hippocampal LTP was unaffected in both genotypes. Similarly, both basic properties of dopaminergic nigral neurons and their responses to D2R activation were normal. These results provide evidence for a regional specificity of the electrophysiological abnormalities observed and demonstrate the reproducibility of such alterations in distinct models of DYT1 dystonia.

Aberrant striatal synaptic plasticity in monogenic parkinsonisms.

In the recent past, the pathogenesis of Parkinson's disease (PD) has evolved from a neurodegenerative disorder considered entirely sporadic to a disease with an unequivocal genetic component. Indeed, different inherited forms of PD have been discovered and characterized, although the functional roles of the gene products identified are still under intense investigation. To gain a better understanding of the cellular and molecular pathogenic mechanisms of hereditary forms of PD, different animal models have been generated. Although most of the rodent models display neither obvious behavioral impairment nor evidence for neurodegeneration, remarkable abnormalities of dopamine-mediated neurotransmission and corticostriatal synaptic plasticity have been described, indicative of a fundamental distortion of network function within the basal ganglia. The picture emerging from a critical review of recent data on monogenic parkinsonisms suggests that mutations in PD genes might cause developmental rearrangements in the corticobasal ganglia circuitry, compensating the dopaminergic dysfunction observed both in mice and humans, in order to maintain proper motor function.

Altered profile and D2-dopamine receptor modulation of high voltage-activated calcium current in striatal medium spiny neurons from animal models of Parkinson's disease.

In the present work we analyzed the profile of high voltage-activated (HVA) calcium (Ca2+) currents in freshly isolated striatal medium spiny neurons (MSNs) from rodent models of both idiopathic and familial forms of Parkinson's disease (PD). MSNs were recorded from reserpine-treated and 6-hydroxydopamine (6-OHDA)-lesioned rats, and from DJ-1 and PINK1 (PTEN induced kinase 1) knockout (-/-) mice. Our analysis showed no significant changes in total HVA Ca2+ current. However, we recorded a net increase in the L-type fraction of HVA Ca2+ current in dopamine-depleted rats, and of both N- and P-type components in DJ-1-/- mice, whereas no significant change in Ca2+ current profile was observed in PINK1-/- mice. Dopamine modulates HVA Ca2+ channels in MSNs, thus we also analyzed the effect of D1 and D2 receptor activation. The effect of the D1 receptor agonist SKF 83822 on Ca2+ current was not significantly different among MSNs from control animals or PD models. However, in both dopamine-depleted rats and DJ-1-/- mice the D2 receptor agonist quinpirole inhibited a greater fraction of HVA Ca2+ current than in the respective controls. Conversely, in MSNs from PINK1-/- mice we did not observe alterations in the effect of D2 receptor activation. Additionally, in both reserpine-treated and 6-OHDA-lesioned rats, the effect of quinpirole was occluded by the selective L-type Ca2+ channel blocker nifedipine, while in DJ-1-/- mice it was mostly occluded by ω-conotoxin GVIA, blocker of N-type channels. These results demonstrate that both dopamine depletion and DJ-1 deletion induce a rearrangement in the HVA Ca2+ channel profile, specifically involving those channels that are selectively modulated by D2 receptors.

Enhanced sensitivity to group II mGlu receptor activation at corticostriatal synapses in mice lacking the familial parkinsonism-linked genes PINK1 or Parkin.

An altered glutamatergic input at corticostriatal synapses has been shown in experimental models of Parkinson's disease (PD). In the present work, we analyzed the membrane and synaptic responses of striatal neurons to metabotropic glutamate (mGlu) receptor activation in two different mouse models of inherited PD, linked to mutations in PINK1 or Parkin genes. Both in PINK1 and Parkin knockout ((-/-)) mice, activation of group I mGlu receptors by 3,5-DHPG caused a membrane depolarization coupled to an increase in firing frequency in striatal cholinergic interneurons that was comparable to the response observed in the respective wild-type (WT) interneurons. The sensitivity to group II and III mGlu receptors was tested on cortically-evoked excitatory postsynaptic potentials (EPSPs) recorded from medium spiny neurons (MSNs). Both LY379268 and L-AP4, agonists for group II and III, respectively, had no effect on intrinsic membrane properties, but dose-dependently reduced the amplitude of corticostriatal EPSPs. However, both in PINK1(-/-) and Parkin(-/-) mice, LY379268, but not L-AP4, exhibited a greater potency as compared to WT in depressing EPSP amplitude. Accordingly, the dose-response curve for the response to LY379268 in both knockout mice was shifted leftward. Moreover, consistent with a presynaptic site of action, both LY379268 and L-AP4 increased the paired-pulse ratio either in PINK1(-/-) and Parkin(-/-) or in WT mice. Acute pretreatment with L-dopa did not rescue the enhanced sensitivity to LY379268. Together, these results suggest that the selective increase in sensitivity of striatal group II mGlu receptors represents an adaptive change in mice in which an altered dopamine metabolism has been documented.

Antiepileptic drugs on calcium currents recorded from cortical and PAG neurons: therapeutic implications for migraine.

Cortex and periaqueductal grey (PAG) play a major role in the pathophysiology of migraine. Some antiepileptic drugs (AEDs) influence the activity of these structures by modulating high-voltage-activated (HVA) Ca(2+) channels and are effective in migraine prevention. The aim of the present study was to investigate the expression of total HVA Ca(2+) channels in cortical and PAG neurons and to study the differential action of AEDs on these channels. Isolated neurons were visually identified based on morphological criteria. HVA currents were recorded by whole-cell patch-clamp technique. The distribution ratio of L-, N-, P-, Q- and R-type HVA Ca(2+) channels was different between cortical and PAG neurons. In particular, we found that P- and Q-type HVA Ca(2+) channels were more expressed in PAG neurons than in cortical cells, whereas L- and R-type HVA Ca(2+) channels showed an opposite distribution. Interestingly, N-type HVA Ca(2+) channels were equally distributed in these two neuronal populations. A differential sensitivity to AEDs of HVA Ca(2+) channels located on cortical and PAG neurons was observed for topiramate (TPM), but not for lamotrigine (LTG) or levetiracetam (LEV). In fact, whereas both LTG and LEV were equally effective and potent in inhibiting HVA Ca(2+) currents in the two neuronal populations, TPM showed a much higher potency and efficacy in blocking these currents in PAG neurons than in cortical pyramidal cells. TPM, in fact, inhibited N-, P- and L-type channels in PAG neurons, whereas in cortical neurons this AED modulated only P- and L-type channels. Unlike the other AEDs investigated, valproic acid did not affect HVA Ca(2+) currents in cortical and PAG neurons. The negative modulation of specific subtypes of HVA Ca(2+) channels by various AEDs can restore normal electrical activity in target brain areas such as cortex and PAG, providing interesting therapeutic approaches in migraine prevention.

Distinct roles of group I mGlu receptors in striatal function.

In the recent past, evidence accumulated in favour of a central role of group I metabotropic glutamate (mGlu) receptors, mGlu1 and mGlu5, in the modulation of cell excitability both of striatal medium spiny projection neurons (MSNs) and interneuronal population. Electrophysiological and pharmacological studies have clearly shown that activation of mGlu1 and mGlu5 receptors exerts distinct actions, depending on the neuronal subtype involved. MGlu5 receptor activation mediates the potentiation of NMDA responses in MSNs, and underlies the retrograde inhibitory signaling by endocannabinoids at corticostriatal synapses. Conversely, both group I mGlu receptors are involved in long-term synaptic plasticity of MSNs. Likewise, either mGlu1 or mGlu5 receptors are engaged in shaping the excitability of large cholinergic interneurons, playing different roles in the membrane responses. Differently, although GABAergic parvalbumin-positive, fast-spiking interneurons have been shown to express both group I receptors, only mGlu1 receptor seems to mediate membrane and synaptic responses. This review provides a brief survey of the cellular and synaptic actions of group I mGlu receptors, and discusses the potential relevance of these findings in neostriatal function and motor control.

Age-related functional changes of high-voltage-activated calcium channels in different neuronal subtypes of mouse striatum.

By means of whole-cell patch-clamp recordings, we characterized the developmental profile of high-voltage-activated (HVA) calcium (Ca(2+)) channel subtypes in distinct neuronal populations of mouse striatum. Acutely dissociated medium spiny neurons (MSNs) and cholinergic interneurons (ChIs) were recorded from mice at five developmental stages: postnatal-days (PD) 14, 23, 40, 150 and 270. During ageing, total HVA Ca(2+) current recorded from both MSNs and ChIs was unchanged. However, the pharmacological analysis of the differential contribution of HVA Ca(2+) channel subtypes showed a significant rearrangement of each component. In both neuronal subtypes, a large fraction of the total HVA current recorded from PD14 mice was inhibited by the L-type HVA channel blocker nifedipine. This dihydropyridine-sensitive component accounted for nearly 50%, in MSNs, and 35%, in ChIs, of total current at PD14, but its contribution was down-regulated up to 20-25% at 9 months. Likewise, the N-type, omega-conotoxin GVIA-sensitive component decreased from 35% to 40% to about 25% in MSNs and 15% in ChIs. The P-type, omega-agatoxin-sensitive fraction did not show significant changes in both neuronal subtypes, whereas the Q-type, omega-conotoxin MVIIC-sensitive channels did show a significant up-regulation at 9 months. As compared with striatal neurons, we recorded pyramidal neurons dissociated from cortical layers IV-V and found no significant developmental change in the different components of HVA Ca(2+) currents. In conclusion, our data demonstrate a functional reconfiguration of HVA Ca(2+) channels in striatal but not cortical pyramidal neurons during mouse development. Such changes might have profound implications for physiological and pathophysiological processes of the striatum.

Altered responses to dopaminergic D2 receptor activation and N-type calcium currents in striatal cholinergic interneurons in a mouse model of DYT1 dystonia.

Early-onset torsion dystonia (DYT1) is an autosomal dominant disease caused by a deletion in the gene encoding the protein torsinA. Recently, a transgenic mouse model of DYT1 has been described, expressing either the human wild-type torsinA (hWT) or mutant torsinA (hMT). We recorded the activity of striatal cholinergic interneurons of hWT, hMT, and control mice. In slice preparations, no significant differences were observed in resting membrane potential (RMP), firing activity, action potential duration or Ih current. Quinpirole, a D2-like dopamine receptor agonist, did not produce detectable effects on RMP of cholinergic interneurons in control mice and hWT mice, but in the hMT mice caused membrane depolarization and an increase in the firing rate. D2 receptor activation inhibits N-type high-voltage-activated calcium currents. We found that, in isolated interneurons from hMT mice, the quinpirole-mediated inhibition of N-type currents was significantly larger than in hWT and controls. Moreover, the N-type component was significantly over-represented in hMT mice. The altered sensitivity of N-type channels in hMT mice could account for the paradoxical excitatory effect of D2 stimulation. Our data support the existence of an imbalance between striatal dopaminergic and cholinergic signaling in DYT1 dystonia.

Enhanced sensitivity of DJ-1-deficient dopaminergic neurons to energy metabolism impairment: role of Na+/K+ ATPase.

DJ-1 gene mutations lead to an inherited form of early-onset parkinsonism. The function of DJ-1 is unclear, though a neuroprotective role has been postulated. Electrophysiological recordings were made of striatal and dopaminergic nigral neurons both of wild-type (WT) and DJ-1-knockout (DJ-1(-/-)) mice. We assessed the responses of dopaminergic cells to combined oxygen and glucose deprivation (OGD), and to the mitochondrial toxin rotenone. OGD induced a membrane hyperpolarization in nigral neurons from WT mice. Similarly, rotenone hyperpolarized neurons and then a depolarization occurred. In DJ-1(-/-) mice, the OGD-induced hyperpolarization was significantly enhanced. Moreover, rotenone caused a shorter hyperpolarization followed by an irreversible depolarization. To evaluate the involvement of Na+/K+ ATPase, we tested ouabain, a Na+/K+ ATPase inhibitor, on two distinct neuronal subtypes. Compared to WT mice, in dopaminergic neurons from DJ-1(-/-) mice, ouabain induced rapid and irreversible membrane potential changes. Notably, this effect was observed at concentrations that were unable to produce membrane potential shifts on striatal spiny neurons, both from WT and DJ-1(-/-) mice. These findings suggest that DJ-1 loss-of-function enhances vulnerability to energy metabolism alterations, and that nigral neurons are particularly sensitive to Na+/K+ ATPase impairment.

Mitochondrial toxins in Basal Ganglia disorders: from animal models to therapeutic strategies.

Current knowledge of the pathogenesis of basal ganglia disorders, such as Huntington's disease (HD) and Parkinson's disease (PD) appoints a central role to a dysfunction in mitochondrial metabolism. The development of animal models, based upon the use of mitochondrial toxins has been successfully introduced to reproduce human disease, leading to important acquisitions. Most notably, experimental evidence supports the existence, within basal ganglia, of a peculiar regional vulnerability to distinct mitochondrial toxins. MPTP and rotenone, both selective inhibitors of mitochondrial complex I have been extensively used to mimic PD. Accordingly, in human PD, a specific dysfunction of complex I activity was found in vulnerable dopaminergic neurons of the substantia nigra. Conversely, in HD a selective impairment of mitochondrial succinate dehydrogenase, key enzyme in complex II activity was found in medium spiny neurons of the caudate-putamen. The relevance of such finding is further demonstrated by the evidence that toxins able to primarily target mitochondrial complex II, such as malonic acid and 3-nitropropionic acid (3-NP), strikingly reproduce the main phenotypic and pathological features of HD.Despite the advances obtained from these experimental models, a deeper understanding of the molecular and cellular mechanisms underlying such neuronal vulnerability is lacking.The present review provides a brief survey of currently utilized animal models of mitochondrial intoxication, in attempt to address the cellular mechanisms triggered by energy metabolism failure and to identify potential therapeutic targets.