Conference Agenda

Historically, the idea that α-synuclein aggregation is responsible for Parkinson's disease (PD) originated about 25 years ago with the discovery of the SNCA gene. In that same year, α-synuclein (α-syn) was identified as a major component of Lewy bodies. Subsequent discoveries of SNCA gene duplications and triplications in individuals with PD supported the ‘gain-of-function’ hypothesis that higher levels of the SNCA protein correlate with increased risk and more severe clinical phenotypes. For years, the paradigm of toxic proteinopathy has served as the framework for understanding neurodegenerative diseases, like PD and Alzheimer's disease. This gain-of-function model posits that proteins, when transformed into amyloid forms, become detrimental, suggesting that reducing these protein levels could yield clinical benefits. Current concepts in PD pathophysiology assert that the spread of α-syn from the lower brainstem is both necessary and sufficient for the onset of PD.

However, recent clinicopathological studies in PD indicate that only 30% of brains with Lewy pathology manifest a neurodegenerative disorder, and neither the distribution nor the load of α-syn pathology reliably permits a postmortem diagnosis of parkinsonism or cognitive impairment. This evidence suggests that α-syn-positive structures are not definitive markers of neuronal dysfunction and degeneration. Conversely, neurodegeneration and cellular dysfunction precede α-syn pathology in the substantia nigra. According to data from three large, community-based longitudinal cohort studies, PD pathology is equally prevalent in subjects with and without parkinsonism.

Taken together, these findings appear to support an alternative ‘loss-of-function’ 'Proteinopenia' paradigm, suggesting that PD may not be due to abnormal α-syn accumulation but rather to the loss of soluble, monomeric α-syn. Consequently, a paradigm shift from the ‘Proteinopathy’ paradigm to the ‘Proteinopenia’ paradigm has been recently advocated to refocus disease-modifying efforts from aggregated protein clearance to soluble (non-aggregating) protein replacement.

Synaptic α-synuclein micro-aggregation constitutes a discerning facet of synucleinopathies such as Parkinson’s disease (PD). As α-synuclein physiologically modulates several synaptic proteins and synaptic vesicle homeostasis, its pathological deposition at nerve terminals severely affects neuronal function and resilience, initiating a retrograde synapse-to-cell body degeneration.

Consistently, we found that several synaptic proteins can be detected in α-synuclein aggregates or can be progressively redistributed following α-synuclein pathological deposition, thus affecting synaptic function. Among them, the phosphoprotein synapsin III (Syn III), a key interactor of α-synuclein in the modulation of dopamine release, is another key component of α-synuclein insoluble fibrils extracted from post-mortem PD brains. Notably, Syn III knock-out mice do not develop α-synuclein fibrillary aggregates, synaptic derangement and dopaminergic neuron degeneration following the injection of adeno-associated viral vectors (AAV) overexpressing human wild type α-synuclein in the substantia nigra, supporting that Syn III controls α-synuclein deposition. Moreover, AAV-based Syn III gene silencing in an α-synuclein transgenic mice at a pathological stage with marked α-synuclein fibrillary aggregation, severe synaptic damage and dysfunction, reduced α-synuclein aggregates, restored dopamine release and prevented the onset of striatal dopaminergic terminal deafferentation and motor deficits. Finally, small molecules disrupting the pathological α-synuclein/Syn III interaction, can reduce α-synuclein synaptic microaggregates and protect dopaminergic neurons from neurodegeneration in mice transgenic for human α-synuclein.

These data support that synaptic α-synuclein microaggregates are the main engine of neuronal degeneration in PD and other synucleinopathies and that Syn III, along with other synaptic proteins, may constitute a suitable therapeutic target for these neurodegenerative disorders.

The main neuropathological hallmarks of Parkinson’s disease (PD) are Lewy bodies (LBs), characterized by α-Synuclein aggregates. The common paradigm that PD is a synucleinopathy relies on the fact that LBs diffusion is correlated with clinical features and α-Synuclein mutations characterize some familial forms of PD.

The failure of clinical trials aimed to reduce α-Synuclein burden, both directly (anti-α-Synuclein antibodies) and indirectly (enhancing the autophagic degradation), together with the lower levels of α-Synuclein aggregates detected in the CSF of PD patients, suggested the idea that synucleinopenia could be the real cause of the pathology.

Thus, many questions arise. PD is driven by synucleinopathy or synucleinopenia? α-Synuclein aggregation is the cause, a consequence or just an epiphenomenon of the disease? The answers to these questions are important for understanding PD etiology, and, consequently, for designing the correct therapeutical approach.

We hypothesized that α-Synuclein aggregation could be due to changes in its interplay with Tubulin/microtubules that are emerging as α-Synuclein interactors also at the synaptic level, where α-Synuclein exerts its main functions. We investigated α-Synuclein oligomers, the earliest aggregated forms, and acetylated α-Tubulin in post-mortem human brain and in the peripheral nervous system using skin biopsy.

High-resolution and 3D reconstruction analysis linked acetylated α-Tubulin redistribution to α-Synuclein oligomerization, leading us to propose a model for LBs morphogenesis. Finally, for the first time in post-mortem human brain, we observed α-Synuclein oligomers in nanotube-like structures, associated with acetylated α-Tubulin enriched neurons, suggesting the prion-like behavior of α-Synuclein. Furthermore, in skin biopsies obtained from PD patients, we observed an increase in α-Synuclein oligomers and a decrease in acetylated α-Tubulin. In conclusion, all these data support our hypothesis that their interplay is altered in PD, indicating the role of acetylated α-Tubulin in α-Synuclein aggregation and providing clues to design novel therapeutic interventions.

Parkinson’s disease (PD) presents the selective loss of A9 dopaminergic (DA) neurons of Substantia Nigra pars compacta (SNpc) and the presence of intracellular aggregates called Lewy bodies. α-synuclein (α-syn) species truncated at the carboxy-terminal (C-terminal) accumulate in pathological inclusions and promote α-syn aggregation and toxicity. Haemoglobin (Hb) is the major oxygen carrier protein in erythrocytes. In addition, Hb is expressed in A9 DA neurons where it influences mitochondrial activity. Hb overexpression increases cells’ vulnerability in a neurochemical model of PD in vitro and forms cytoplasmic and nucleolar aggregates upon short-term overexpression in mouse SNpc. In this study, α and β-globin chains were co-expressed in DA cells of SNpc in vivo upon stereotaxic injections of an Adeno-Associated Virus isotype 9 (AAV9) and in DA iMN9D cells in vitro. Long-term Hb over-expression in SNpc induced the loss of about 50% of DA neurons, mild motor impairments, and deficits in recognition and spatial working memory. Hb triggered the formation of endogenous α-syn C-terminal truncated species. Similar α-syn fragments were found in vitro in DA iMN9D cells over-expressing α and β- globins when treated with pre-formed α-syn fibrils. Our study positions Hb as a relevant player in PD pathogenesis for its ability to trigger DA cells’ loss in vivo and the formation of C-terminal α-syn fragments.