Conference Agenda

Pathological deposition of α-synuclein (α-syn) microaggregates at synaptic terminals characterizes the early prodromal phases of Parkinson’s disease (PD). This phenomenon may trigger a retrograde synapse-to-cell-body degeneration, but we still ignore how the deposition of α-syn microaggregates can gradually affect nigrostriatal synapses to drive neuronal loss.

We used mice overexpressing human truncated (1-120) α-syn on the C57BL/6JOlaHsd α-syn null background (SYN120 tg), exhibiting extensive deposition of α-syn fibrillary microaggregates in nigrostriatal terminals and dopamine release deficits at 12 months of age, to stage striatal dopaminergic terminal degeneration, dopamine release dysfunction as well as synaptic markers alterations by two-photon and confocal microscopy and in vivo microdialysis. 8, 10 and 12 month-old SYN120 tg, C57BL6JolaHsd wt and C57BL/6J (expressing endogenous mouse α-syn) control mice were analyzed.

Early loss of vesicular monoamine transporter 2 (VMAT2) without dopamine release deficits or alterations of tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunolabeling was observed in 8- and 10-month-old SYN120 tg mice compared to C57BL/6J animals. Later, 12-month-old SYN120 tg mice showed dopamine release drop and dopamine turnover perturbation accompanied by a paradoxical dysfunctional DAT increase confirmed by vertical microdialysis and a patchy striatal fibers deafferentation detected by 3D two-photon microscopy of whole dorsal striata. This was associated with a reduced number of contacts between striatal TH-positive fibers and choline acetyltransferase (ChAT)-positive striatal neurons in 12-month-old SYN120 tg mice.

These data support that the progressive accumulation of α-syn at nigrostriatal terminals in the prodromal stages of PD produces a very early loss of VMAT2 that anticipates the synaptic dopamine release deficits. Moreover, they support that the initial phase of pathological α-syn-induced dopaminergic dysfunction is characterized by dopamine turnover perturbation with accumulation of dysfunctional DAT and that these pre-degenerative synaptic alterations can initiate nigrostriatal deafferentation, reflected by a reduction in the synaptic contacts established with cholinergic neurons.

Impairment of synaptic activity is a hallmark of movement disorders such as dystonia. In particular, DYT1 dystonia is characterized by reduced penetrance and several endophenotypes converging on synaptic dysfunction have been shown in different experimental models. Intriguingly, torsinA (TA), the protein causative of DYT1 dystonia, has been found to interact with alpha-synuclein (α-Syn). Both proteins act as molecular chaperones and control synaptic machinery. Despite such evidence, the role of α-Syn in dystonia has never been investigated. We explored whether α-Syn and N-ethylmaleimide sensitive fusion attachment protein receptor proteins (SNAREs), that are known to be modulated by α-Syn, may be involved in DYT1 dystonia synaptic dysfunction. We used electrophysiological and biochemical techniques to study synaptic alterations in the dorsal striatum of the Tor1a^+/Δgag mouse model of DYT1 dystonia. In the Tor1a^+/Δgag DYT1 mutant mice, we found a significant reduction of α-Syn levels in whole striata, mainly involving glutamatergic corticostriatal terminals. Strikingly, the striatal levels of the vesicular SNARE VAMP-2, a direct α-Syn interactor, and of the transmembrane SNARE synaptosome-associated protein 23 (SNAP-23), that promotes glutamate synaptic vesicles release, were markedly decreased in mutant mice. Moreover, we detected an impairment of miniature glutamatergic postsynaptic currents (mEPSCs) recorded from striatal spiny neurons, in parallel with a robust alteration in release probability. Finally, we also observed a significant reduction of TA striatal expression in α-Syn null mice. Our data demonstrate an unprecedented relationship between TA and α-Syn, and reveal that α-Syn and SNAREs alterations characterize the synaptic dysfunction underlying DYT1 dystonia.

Movement disorders comprise a group of heterogeneous diseases with often complex clinical phenotypes.A phenotypic overlap between dystonia, chorea, ataxia, epilepsy, and neurodevelopmental disorders is becoming increasingly evident.Overlapping symptoms and a lack of diagnostic biomarkers may impede making a definitive diagnosis.

Recently diagnosed chorea and dystonia causing genes has allowed the recognition of converging molecular pathways of basal ganglia circuits.

In this presentation I will highlight the genetic findings and disease mechanisms that characterize these genetic postsynaptic movement disorders.

Particularly a dysregulation of cAMP signaling in the striato-nigral and striato-pallidal neurons have been identified as causes of hyperkinetic movement disorders.

Predominantly ADCY5 gene, encoding for an adenylyl cyclases mediate G protein-coupled receptor signaling through the synthesis of the second messenger cAMP, has been associated with infantile onset chorea, dystonia and myoclonus.

Regulation G proteins is also critical in coordinated movement. Mutations in GNAO1, GNB1 and GNAL genes encoding respectively for the G protein subunit alpha o1, the G protein subunit beta 1 and the G protein subunit alpha, cause complex hyperkinetic movement disorders. Furthermore, an aberrant function of the purine metabolic enzyme HPRT or the G-protein coupled receptor 88 (GPR88) are also related to a genetic origin of chorea.

The compartmentalization of PDEs shapes the local gradients of cyclic nucleotides in the striatum, which are essential for the regulation of the motor function. Accordingly, several pathogenic substitutions in these cAMP degradative enzymes induce movement disorders, such as PDE2A, PDE8B and PDE10A.

NMDAR gene family (GRIN) are responsible for pediatric encephalopathies leading to intellectual disability, hypotonia, communication deficits, motor impairment and movement disorder.

Despite these genetic progress disease-specific personalized treatments is still not currently available. Precision medicine approaches, targeting the specific gene defect and specific post synaptic pathways, may provide a revolutionize strategy to treat these severe genetic movement disorders.

The SNCA gene encodes for alpha-synuclein (alpha-syn), a protein that is widely renowned for its role in synaptogenesis and implication in both Parkinson’s Disease (PD) and aging, but research efforts are needed to clarify its physiological functions. SNCA-AS1, the antisense transcript to the SNCA gene, has been found up-regulated in patients affected by sporadic PD and its transcriptional modification is implicated in synapse-related genes expression. This work aims to report how SNCA-AS1 affects both synaptic processes and alpha-syn’s expression. SH-SY5Y cells stably transfected with SNCA-AS1 to overexpress the gene, whereas the knock-down model was performed via CRISPR-dCas9. Real Time-PCR and western blot were used to verify SNCA-AS1’s effects on SNCA’s expression and synapses-related markers. SNCA half-life was assessed via Actinomycin D treatment. Alpha-syn’s expression was evaluated in presence of Cycloheximide. TEM and SEM were performed to analyze morphological aspects of synapses during the neural differentiation. RNA-pulldown studies were performed to assess SNCA-AS1 interactome. The overexpression of SNCA-AS1 in SH-SY5Y cells upregulates both alpha-syn’s mRNA and protein, whilst its knock-down decreases both targets. This strongly impacts on neurites’ extension and synapses’ morphological ultrastructural aspect, as assessed by SEM and TEM. Through specific molecular signatures SNCA-AS1 causes transcriptional modification in synaptic modulation pathways recapitulating the aging-related decline, as confirmed by RNA-Seq studies. RNA-pulldown studies highlighted interactions of the antisense with genes encoding for synaptic processes, suggesting its deep involvement in the process. Moreover, we demonstrate that the SNCA-AS1 overexpression reduces the half-life of SNCA mRNA, contrary to what happens in the knock-down model. Lastly, we report that SNCA-AS1 overexpressing cells release alpha-syn in the medium. Our results show that SNCA-AS1 impacts alpha-syn expression, synapses biology and PD-related genes.