La disfunzione del recettore striatale D2 induce un’alterata trasmissione GABAergica in un modello murino di distonia DYT1

La distonia DYT1 è una grave forma di distonia generalizzata causata da una mutazione del gene DYT1 che codifica per la proteina TorsinA. La funzione di tale proteina rimane ancora poco chiara anche se è stato proposto che possa svolgere importanti funzioni nel traffico proteico intracellulare e nei processi secretori. Lo striato, all'interno dei gangli della base svolge un importante ruolo nella regolazione dell'attività motoria, ed alterazioni a carico di tale struttura appaiono essere coinvolti nella patogenesi della distonia. Ho registrato pertanto le correnti sinaptiche spontanee sia di tipo GABAergico che glutamatergico in neuroni spinosi striatali (MSNs) da animali che sovraesprimevano la proteina umana mutata (hMT) confrontandoli poi con animali di controllo (CTRL) e con quelli che esprimevano la proteina umana non-mutata (hWT). Gli animali mutati presentavano un significativo aumento nella frequenza degli eventi sinaptici GABAergici (sIPSCs) non accompagnato però da variazioni nell'ampiezza di tali correnti. Al contrario l'attività spontanea di tipo glutamatergico (sEPSC) risultava essere del tutto normale. L'inibizione GABAergica striatale è di origine esclusivamente instrinseca e deriva da due distinte fonti. Una delle più importanti tuttavia fa capo agli interneuroni GABAergici Fast Spiking (FS). Ho pertanto verificato l'ipotesi che tali cellule potessero presentare alterazioni nella loro normale funzionalità. Sia gli sIPSCs che gli sEPSC registrati risultavano tuttavia essere invariati fra gli animali hMT, hWT e quelli di controllo. In condizioni fisiologiche l'attivazione del recettore dopaminergico D2 agisce presinapticamente inibendo il rilascio di GABA. Nei MSNs di animali di controllo e hWT, tale funzionalità risultava essere del tutto preservata. L'applicazione di quinpirolo (agonista D2-like) portava infatti ad una significativa riduzione della frequenza degli sIPSCs misurati. Tale effetto tuttavia era assente negli animali hMT. Inoltre sia MSNs sia FS di topi hMT non presentavano l'effetto inibitorio tipico del quinpirolo sulle correnti sinaptiche evocate tramite stimolazione elettrica (eIPSCs). In conclusione il mio lavoro dimostra la presenza di un'alterata attività del circuito GABAergico striatale in un modello animale di distonia DYT1, che può essere in parte giustificata da una disfunzione del recettore dopaminergico D2.DYT1 dystonia is a severe form of inherited generalized dystonia, caused by a deletion in the DYT1 gene encoding the protein torsinA. The physiological function of torsinA is unclear, though it has been proposed to perform chaperone-like functions, assist in protein trafficking, membrane fusion and participate in secretory processing. Alterations in GABAergic signaling have been involved in the pathogenesis of dystonia. I recorded GABA- and glutamate-mediated synaptic currents from striatal neurons obtained from a mouse model of DYT1 dystonia. In medium spiny neurons (MSNs) from mice expressing human mutant torsinA (hMT), we observed a significantly higher frequency, but not amplitude, of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature currents (mIPSCs), whereas glutamatergic spontaneous excitatory synaptic potentials (sEPSCs) activity was normal. No alterations were found in mice overexpressing normal human torsinA (hWT). To identify the possible sources of the increased GABAergic tone, I recorded GABAergic Fast-Spiking (FS) interneurons that exert a feed-forward inhibition on MSNs. Both sEPSC and sIPSC recorded from hMT FS interneurons were comparable to hWT and controls.In physiological conditions, dopamine (DA) D2 receptor act presynaptically to reduce striatal GABA release. Notably, application of the D2-like receptor agonist quinpirole failed to reduce the frequency of sIPSCs in MSNs from hMT as compared to hWT and controls. Likewise, the inhibitory effect of quinpirole was lost on evoked IPSCs both in MSNs and FS interneurons from hMT mice. My findings demonstrate a disinhibition of GABAergic synaptic activity, that can be partially attributed to a D2 DA receptor dysregulation. A rise in GABA transmission would result in a profound alteration of striatal output, that might be relevant to the pathogenesis of dystonia

Optogenetic Activation of Striatopallidal Neurons Reveals Altered HCN Gating in DYT1 Dystonia

Summary: Firing activity of external globus pallidus (GPe) is crucial for motor control and is severely perturbed in dystonia, a movement disorder characterized by involuntary, repetitive muscle contractions. Here, we show that GPe projection neurons exhibit a reduction of firing frequency and an irregular pattern in a DYT1 dystonia model. Optogenetic activation of the striatopallidal pathway fails to reset pacemaking activity of GPe neurons in mutant mice. Abnormal firing is paralleled by alterations in motor learning. We find that loss of dopamine D2 receptor-dependent inhibition causes increased GABA input at striatopallidal synapses, with subsequent downregulation of hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels. Accordingly, enhancing in vivo HCN channel activity or blocking GABA release restores both the ability of striatopallidal inputs to pause ongoing GPe activity and motor coordination deficits. Our findings demonstrate an impaired striatopallidal connectivity, supporting the central role of GPe in motor control and, more importantly, identifying potential pharmacological targets for dystonia

Impaired dopamine- and adenosine-mediated signaling and plasticity in a novel rodent model for DYT25 dystonia

Abstract Dystonia is a neurological movement disorder characterized by sustained or intermittent involuntary muscle contractions. Loss-of-function mutations in the GNAL gene have been identified to be the cause of "isolated" dystonia DYT25. The GNAL gene encodes for the guanine nucleotide-binding protein G(olf) subunit alpha (Gαolf), which is mainly expressed in the olfactory bulb and the striatum and functions as a modulator during neurotransmission coupling with D1R and A2AR. Previously, heterozygous Gαolf -deficient mice (Gnal+/−) have been generated and showed a mild phenotype at basal condition. In contrast, homozygous deletion of Gnal in mice (Gnal−/−) resulted in a significantly reduced survival rate. In this study, using the CRISPR-Cas9 system we generated and characterized heterozygous Gnal knockout rats (Gnal+/−) with a 13 base pair deletion in the first exon of the rat Gnal splicing variant 2, a major isoform in both human and rat striatum. Gnal+/− rats showed early-onset phenotypes associated with impaired dopamine transmission, including reduction in locomotor activity, deficits in rotarod performance and an abnormal motor skill learning ability. At cellular and molecular level, we found down-regulated Arc expression, increased cell surface distribution of AMPA receptors, and the loss of D2R-dependent corticostriatal long-term depression (LTD) in Gnal+/− rats. Based on the evidence that D2R activity is normally inhibited by adenosine A2ARs, co-localized on the same population of striatal neurons, we show that blockade of A2ARs restores physiological LTD. This animal model may be a valuable tool for investigating Gαolf function and finding a suitable treatment for dystonia associated with deficient dopamine transmission

La disfunzione del recettore striatale D2 induce un’alterata trasmissione GABAergica in un modello murino di distonia DYT1

La distonia DYT1 è una grave forma di distonia generalizzata causata da una mutazione del gene DYT1 che codifica per la proteina TorsinA. La funzione di tale proteina rimane ancora poco chiara anche se è stato proposto che possa svolgere importanti funzioni nel traffico proteico intracellulare e nei processi secretori. Lo striato, all'interno dei gangli della base svolge un importante ruolo nella regolazione dell'attività motoria, ed alterazioni a carico di tale struttura appaiono essere coinvolti nella patogenesi della distonia. Ho registrato pertanto le correnti sinaptiche spontanee sia di tipo GABAergico che glutamatergico in neuroni spinosi striatali (MSNs) da animali che sovraesprimevano la proteina umana mutata (hMT) confrontandoli poi con animali di controllo (CTRL) e con quelli che esprimevano la proteina umana non-mutata (hWT). Gli animali mutati presentavano un significativo aumento nella frequenza degli eventi sinaptici GABAergici (sIPSCs) non accompagnato però da variazioni nell'ampiezza di tali correnti. Al contrario l'attività spontanea di tipo glutamatergico (sEPSC) risultava essere del tutto normale. L'inibizione GABAergica striatale è di origine esclusivamente instrinseca e deriva da due distinte fonti. Una delle più importanti tuttavia fa capo agli interneuroni GABAergici Fast Spiking (FS). Ho pertanto verificato l'ipotesi che tali cellule potessero presentare alterazioni nella loro normale funzionalità. Sia gli sIPSCs che gli sEPSC registrati risultavano tuttavia essere invariati fra gli animali hMT, hWT e quelli di controllo. In condizioni fisiologiche l'attivazione del recettore dopaminergico D2 agisce presinapticamente inibendo il rilascio di GABA. Nei MSNs di animali di controllo e hWT, tale funzionalità risultava essere del tutto preservata. L'applicazione di quinpirolo (agonista D2-like) portava infatti ad una significativa riduzione della frequenza degli sIPSCs misurati. Tale effetto tuttavia era assente negli animali hMT. Inoltre sia MSNs sia FS di topi hMT non presentavano l'effetto inibitorio tipico del quinpirolo sulle correnti sinaptiche evocate tramite stimolazione elettrica (eIPSCs). In conclusione il mio lavoro dimostra la presenza di un'alterata attività del circuito GABAergico striatale in un modello animale di distonia DYT1, che può essere in parte giustificata da una disfunzione del recettore dopaminergico D2.DYT1 dystonia is a severe form of inherited generalized dystonia, caused by a deletion in the DYT1 gene encoding the protein torsinA. The physiological function of torsinA is unclear, though it has been proposed to perform chaperone-like functions, assist in protein trafficking, membrane fusion and participate in secretory processing. Alterations in GABAergic signaling have been involved in the pathogenesis of dystonia. I recorded GABA- and glutamate-mediated synaptic currents from striatal neurons obtained from a mouse model of DYT1 dystonia. In medium spiny neurons (MSNs) from mice expressing human mutant torsinA (hMT), we observed a significantly higher frequency, but not amplitude, of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature currents (mIPSCs), whereas glutamatergic spontaneous excitatory synaptic potentials (sEPSCs) activity was normal. No alterations were found in mice overexpressing normal human torsinA (hWT). To identify the possible sources of the increased GABAergic tone, I recorded GABAergic Fast-Spiking (FS) interneurons that exert a feed-forward inhibition on MSNs. Both sEPSC and sIPSC recorded from hMT FS interneurons were comparable to hWT and controls.In physiological conditions, dopamine (DA) D2 receptor act presynaptically to reduce striatal GABA release. Notably, application of the D2-like receptor agonist quinpirole failed to reduce the frequency of sIPSCs in MSNs from hMT as compared to hWT and controls. Likewise, the inhibitory effect of quinpirole was lost on evoked IPSCs both in MSNs and FS interneurons from hMT mice. My findings demonstrate a disinhibition of GABAergic synaptic activity, that can be partially attributed to a D2 DA receptor dysregulation. A rise in GABA transmission would result in a profound alteration of striatal output, that might be relevant to the pathogenesis of dystonia

Managing the Resource Continuum in a Real Video Surveillance Scenario

Over the last years, the number of IoT devices has grown exponentially. Thus, Fog and Edge computing move part of the computation closer to data sources, exploiting interconnected devices in a computing continuum viewpoint. These devices are heterogeneous in terms of performance, features, and capabilities, requiring proper programming models and run-time management layers. This work presents a first version of the BarMan open-source framework. We developed a run-time task allocation policy to maximize application performance and through an experimental evaluation performed on a real cluster, we evaluated different execution scenarios. The results show an improvement up to 66% on the frame processing latency with respect to a monolithic solution

Facemasks and face recognition: Potential impact on synaptic plasticity

Visual recognition of facial expression modulates our social interactions. Compelling experimental evidence indicates that face conveys plenty of information that are fundamental for humans to interact. These are encoded at neural level in specific cortical and subcortical brain regions through activity- and experience-dependent synaptic plasticity processes. The current pandemic, due to the spread of SARS-CoV-2 infection, is causing relevant social and psychological detrimental effects. The institutional recommendations on physical distancing, namely social distancing and wearing of facemasks are effective in reducing the rate of viral spread. However, by impacting social interaction, facemasks might impair the neural responses to recognition of facial cues that are overall critical to our behaviors. In this survey, we briefly review the current knowledge on the neurobiological substrate of facial recognition and discuss how the lack of salient stimuli might impact the ability to retain and consolidate learning and memory phenomena underlying face recognition. Such an "abnormal" visual experience raises the intriguing possibility of a "reset" mechanism, a renewed ability of adult brain to undergo synaptic plasticity adaptations

Endogenous reverse transcriptase as a mediator of ursolic acid's antiproliferative and differentiating effects in human cancer cell lines

Ursolic acid (UA) is a pentacyclic triterpenoid compound that is widely distributed in the plant kingdom and has a broad range of biological effects. Here, we examined the effects of UA on the proliferation and differentiation of human tumor cell lines from melanoma (A375), glioblastoma (U87) and thyroid anaplastic carcinoma (ARO), and on the proliferation of a non-transformed human fibroblast cell line (WI-38). The results show that UA inhibits tumor cell proliferation in a dose- and time-dependent manner. Consistent with this finding, UA treatment promotes differentiation of all of the analyzed tumor cell lines. Interestingly, we found that UA inhibits the endogenous reverse transcriptase (RT) activity in tumor cells, which has recently been shown to be involved in the control of proliferation and differentiation of neoplastic cells. Considering these findings, we suggest that the observed anti-proliferative and differentiating effects of UA may be related to this target

Loss of muscarinic autoreceptor function impairs long-term depression but not long-term potentiation in the striatum

Muscarinic autoreceptors regulate cholinergic tone in the striatum. We investigated the functional consequences of genetic deletion of striatal muscarinic autoreceptors by means of electrophysiological recordings from either medium spiny neurons (MSNs) or cholinergic interneurons (ChIs) in slices from single M(4) or double M(2)/M(4) muscarinic acetylcholine receptor (mAChR) knock-out (−/−) mice. In control ChIs, the muscarinic agonist oxotremorine (300 nm) produced a self-inhibitory outward current that was mostly reduced in M(4)(−/−) and abolished in M(2)/M(4)(−/−) mice, suggesting an involvement of both M(2) and M(4) autoreceptors. In MSNs from both M(4)(−/−) and M(2)/M(4)(−/−) mice, muscarine caused a membrane depolarization that was prevented by the M(1) receptor-preferring antagonist pirenzepine (100 nm), suggesting that M(1) receptor function was unaltered. Acetylcholine has been involved in striatal long-term potentiation (LTP) or long-term depression (LTD) induction. Loss of muscarinic autoreceptor function is predicted to affect synaptic plasticity by modifying striatal cholinergic tone. Indeed, high-frequency stimulation of glutamatergic afferents failed to induce LTD in MSNs from both M(4)(−/−) and M(2)/M(4)(−/−) mice, as well as in wild-type mice pretreated with the M(2)/M(4) antagonist AF-DX384 (11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,1 1-dihydro-6H-pyrido[2,3b][1,4] benzodiazepin-6-one). Interestingly, LTD could be restored by either pirenzepine (100 nm) or hemicholinium-3 (10 μm), a depletor of endogenous ACh. Conversely, LTP induction did not show any difference among the three mouse strains and was prevented by pirenzepine. These results demonstrate that M(2)/M(4) muscarinic autoreceptors regulate ACh release from striatal ChIs. As a consequence, endogenous ACh drives the polarity of bidirectional synaptic plasticity

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

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