Synapsin III gene silencing redeems alpha-synuclein transgenic mice from Parkinson's disease-like phenotype.

Fibrillary aggregated α-synuclein (α-syn) deposition in Lewy bodies (LB) characterizes Parkinson's disease (PD) and is believed to trigger dopaminergic synaptic failure and a retrograde terminal-to-cell body neuronal degeneration. We described that the neuronal phosphoprotein synapsin III (Syn III) cooperates with α-syn to regulate dopamine (DA) release and can be found in the insoluble α-syn fibrils composing LB. Moreover, we showed that α-syn aggregates deposition, and the associated onset of synaptic deficits and neuronal degeneration occurring following adeno-associated viral vectors-mediated overexpression of human α-syn in the nigrostriatal system are hindered in Syn III knock out mice. This supports that Syn III facilitates α-syn aggregation. Here, in an interventional experimental design, we found that by inducing the gene silencing of Syn III in human α-syn transgenic mice at PD-like stage with advanced α-syn aggregation and overt striatal synaptic failure, we could lower α-syn aggregates and striatal fibers loss. In parallel, we observed recovery from synaptic vesicles clumping, DA release failure, and motor functions impairment. This supports that Syn III consolidates α-syn aggregates, while its downregulation enables their reduction and redeems the PD-like phenotype. Strategies targeting Syn III could thus constitute a therapeutic option for PD.

Changes in α-Synuclein Posttranslational Modifications in an AAV-Based Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) pathology is characterized by the loss of dopaminergic neurons of the nigrostriatal system and accumulation of Lewy bodies (LB) and Lewy neurites (LN), inclusions mainly composed of alpha-synuclein (α-Syn) fibrils. Studies linking the occurrence of mutations and multiplications of the α-Syn gene (SNCA) to the onset of PD support that α-Syn deposition may play a causal role in the disease, in line with the hypothesis that disease progression may correlate with the spreading of LB pathology in the brain. Interestingly, LB accumulate posttranslationally modified forms of α-Syn, suggesting that α-Syn posttranslational modifications impinge on α-Syn aggregation and/or toxicity. Here, we aimed at investigating changes in α-Syn phosphorylation, nitration and acetylation in mice subjected to nigral stereotaxic injections of adeno-associated viral vectors inducing overexpression of human α-Syn (AAV-hα-Syn), that model genetic PD with SNCA multiplications. We detected a mild increase of serine (Ser) 129 phosphorylated α-Syn in the substantia nigra (SN) of AAV-hα-Syn-injected mice in spite of the previously described marked accumulation of this PTM in the striatum. Following AAV-hα-Syn injection, tyrosine (Tyr) 125/136 nitrated α-Syn accumulation in the absence of general 3-nitrotirosine (3NT) or nitrated-Tyr39 α-Syn changes and augmented protein acetylation abundantly overlapping with α-Syn immunopositivity were also detected.

Distinct alpha-Synuclein species induced by seeding are selectively cleared by the Lysosome or the Proteasome in neuronally differentiated SH-SY5Y cells.

A major pathological feature of Parkinson's disease (PD) is the aberrant accumulation of misfolded assemblies of alpha-synuclein (α-Syn). Protein clearance appears as a regulator of the 'α-Syn burden' underlying PD pathogenesis. The picture emerging is that a combination of pathways with complementary roles, including the Proteasome System and the Autophagy-Lysosome Pathway, contributes to the intracellular degradation of α-Syn. This study addresses the mechanisms governing the degradation of α-Syn species seeded by exogenous fibrils in neuronally differentiated SH-SY5Y neuroblastoma cells with inducible expression of α-Syn. Using human α-Syn recombinant fibrils (pre-formed fibrils, PFFs), seeding and aggregation of endogenous Proteinase K (PK)-resistant α-Syn species occurs within a time frame of 6 days, and is still prominent after 12 days of PFF addition. Clearance of α-Syn assemblies in this inducible model was enhanced after switching off α-Syn expression with doxycycline. Lysosomal inhibition led to accumulation of SDS-soluble α-Syn aggregates 6 days after PFF-addition or when switching off α-Syn expression. Additionally, the autophagic enhancer, rapamycin, induced the clearance of α-Syn aggregates 13 days post-PFF addition, indicating that autophagy is the major pathway for aggregated α-Syn clearance. SDS-soluble phosphorylated α-Syn at S129 was only apparent at 7 days of incubation with a higher amount of PFFs. Proteasomal inhibition resulted in further accumulation of SDS-soluble phosphorylated α-Syn at S129, with limited PK resistance. Our data suggest that in this inducible model autophagy is mainly responsible for the degradation of fibrillar α-Syn, whereas the proteasome system is responsible, at least in part, for the selective clearance of phosphorylated α-Syn oligomers.

The good and bad of therapeutic strategies that directly target α-synuclein.

Synucleinopathies are neurodegenerative diseases characterized by the accumulation of either neuronal/axonal or glial insoluble proteinaceous aggregates mainly composed of α-synuclein (α-syn). Among them, the most common disorders are Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and some forms of familial parkinsonism. Both α-syn fibrils and oligomers have been found to exert toxic effects on neurons or oligodendroglial cells, can activate neuroinflammatory responses, and mediate the spreading of α-syn pathology. This poses the question of which is the most toxic α-syn species. What is worst, α-syn appears as a very peculiar protein, exerting multiple physiological functions in neurons, especially at synapses, but without acquiring a stable tertiary structure. Its conformation is particularly plastic, and the protein can exist in a natively unfolded state (mainly in solution), partially α-helical folded state (when it interacts with biological membranes), or oligomeric state (tetramers or dimers with debated functional profile). The extent of α-syn expression impinges on the resilience of neuronal cells, as multiplications of its gene locus, or overexpression, can cause neurodegeneration and onset of motor phenotype. For these reasons, one of the main challenges in the field of synucleinopathies, which still nowadays can only be managed by symptomatic therapies, has been the development of strategies aimed at reducing α-syn levels, oligomer formation, fibrillation, or cell-to-cell transmission. This review resumes the therapeutic approaches that have been proposed or are under development to counteract α-syn pathology by direct targeting of this protein and discuss their pros and cons in relation to the current state-of-the-art α-syn biology.

Alpha synuclein post translational modifications: potential targets for Parkinson's disease therapy?

Parkinson's disease (PD) is the most common neurodegenerative disorder with motor symptoms. The neuropathological alterations characterizing the brain of patients with PD include the loss of dopaminergic neurons of the nigrostriatal system and the presence of Lewy bodies (LB), intraneuronal inclusions that are mainly composed of alpha-synuclein (α-Syn) fibrils. The accumulation of α-Syn in insoluble aggregates is a main neuropathological feature in PD and in other neurodegenerative diseases, including LB dementia (LBD) and multiple system atrophy (MSA), which are therefore defined as synucleinopathies. Compelling evidence supports that α-Syn post translational modifications (PTMs) such as phosphorylation, nitration, acetylation, O-GlcNAcylation, glycation, SUMOylation, ubiquitination and C-terminal cleavage, play important roles in the modulation α-Syn aggregation, solubility, turnover and membrane binding. In particular, PTMs can impact on α-Syn conformational state, thus supporting that their modulation can in turn affect α-Syn aggregation and its ability to seed further soluble α-Syn fibrillation. This review focuses on the importance of α-Syn PTMs in PD pathophysiology but also aims at highlighting their general relevance as possible biomarkers and, more importantly, as innovative therapeutic targets for synucleinopathies. In addition, we call attention to the multiple challenges that we still need to face to enable the development of novel therapeutic approaches modulating α-Syn PTMs.

Methylphenidate Analogues as a New Class of Potential Disease-Modifying Agents for Parkinson's Disease: Evidence from Cell Models and Alpha-Synuclein Transgenic Mice.

Parkinson's disease (PD) is characterized by dopaminergic nigrostriatal neurons degeneration and Lewy body pathology, mainly composed of α-synuclein (αSyn) fibrillary aggregates. We recently described that the neuronal phosphoprotein Synapsin III (Syn III) participates in αSyn pathology in PD brains and is a permissive factor for αSyn aggregation. Moreover, we reported that the gene silencing of Syn III in a human αSyn transgenic (tg) mouse model of PD at a pathological stage, manifesting marked insoluble αSyn deposits and dopaminergic striatal synaptic dysfunction, could reduce αSyn aggregates, restore synaptic functions and motor activities and exert neuroprotective effects. Interestingly, we also described that the monoamine reuptake inhibitor methylphenidate (MPH) can recover the motor activity of human αSyn tg mice through a dopamine (DA) transporter-independent mechanism, which relies on the re-establishment of the functional interaction between Syn III and α-helical αSyn. These findings support that the pathological αSyn/Syn III interaction may constitute a therapeutic target for PD. Here, we studied MPH and some of its analogues as modulators of the pathological αSyn/Syn III interaction. We identified 4-methyl derivative I-threo as a lead candidate modulating αSyn/Syn III interaction and having the ability to reduce αSyn aggregation in vitro and to restore the motility of αSyn tg mice in vivo more efficiently than MPH. Our results support that MPH derivatives may represent a novel class of αSyn clearing agents for PD therapy.

An updated reappraisal of synapsins: structure, function and role in neurological and psychiatric disorders.

Synapsins (Syns) are phosphoproteins strongly involved in neuronal development and neurotransmitter release. Three distinct genes SYN1, SYN2 and SYN3, with elevated evolutionary conservation, have been described to encode for Synapsin I, Synapsin II and Synapsin III, respectively. Syns display a series of common features, but also exhibit distinctive localization, expression pattern, post-translational modifications (PTM). These characteristics enable their interaction with other synaptic proteins, membranes and cytoskeletal components, which is essential for the proper execution of their multiple functions in neuronal cells. These include the control of synapse formation and growth, neuron maturation and renewal, as well as synaptic vesicle mobilization, docking, fusion, recycling. Perturbations in the balanced expression of Syns, alterations of their PTM, mutations and polymorphisms of their encoding genes induce severe dysregulations in brain networks functions leading to the onset of psychiatric or neurological disorders. This review presents what we have learned since the discovery of Syn I in 1977, providing the state of the art on Syns structure, function, physiology and involvement in central nervous system disorders.