Molecular characterization of an aggregation-prone variant of alpha-synuclein used to model synucleinopathies

The misfolding and aggregation of alpha-synuclein (aSyn) are thought to be central events in synucleinopathies. The physiological function of aSyn has been related to vesicle binding and trafficking, but the precise molecular mechanisms leading to aSyn pathogenicity are still obscure. In cell models, aSyn does not readily aggregate, even upon overexpression. Therefore, cellular models that enable the study of aSyn aggregation are essential tools for our understanding of the molecular mechanisms that govern such processes. Here, we investigated the structural features of SynT, an artificial variant of aSyn that has been widely used as a model of aggregation in mammalian cell systems, since it is more prone to aggregation than aSyn. Using Nuclear Magnetic Resonance (NMR) spectroscopy we performed a detailed structural characterization of SynT through a systematic comparison with normal, unmodified aSyn. Interestingly, we found that the conformations adopted by SynT resemble those described for the unmodified protein, demonstrating the usefulness of SynT as a model for aSyn aggregation. However, subtle differences were observed at the N-terminal region involving transient intra and/or intermolecular interactions that are known to regulate aSyn aggregation. Importantly, our results indicate that disturbances in the N-terminal region of SynT, and the consequent decrease in membrane binding of the modified protein, might contribute to the observed aggregation behavior of aSyn, and validate the use of SynT, one of the few models of aSyn aggregation in cultured cells

Propagation of tau pathology by alzheimer PHF: seeding of tau isoforms in wild-type mouse and in cells.

info:eu-repo/semantics/nonPublishe

Stereotaxic injection of fibrillar PHF from Alzheimer brain induces propagation of argyrophilic grains constituted of murine tau in mouse brains

info:eu-repo/semantics/publishe

Propagation of tau pathology by Alzheimer PHF: Seeding of tau isoforms in wild-type and mutant tau transgenic mouse brains.

info:eu-repo/semantics/nonPublishe

High-Molecular-Weight Paired Helical Filaments from Alzheimer Brain Induces Seeding of Wild-Type Mouse Tau into an Argyrophilic 4R Tau Pathology in Vivo.

In Alzheimer disease, the development of tau pathology follows neuroanatomically connected pathways, suggesting that abnormal tau species might recruit normal tau by passage from cell to cell. Herein, we analyzed the effect of stereotaxic brain injection of human Alzheimer high-molecular-weight paired helical filaments (PHFs) in the dentate gyrus of wild-type and mutant tau THY-Tau22 mice. After 3 months of incubation, wild-type and THY-Tau22 mice developed an atrophy of the dentate gyrus and a tau pathology characterized by Gallyas and tau-positive grain-like inclusions into granule cells that extended in the hippocampal hilus and eventually away into the alveus, and the fimbria. Gallyas-positive neuropil threads and oligodendroglial coiled bodies were also observed. These tau inclusions were composed only of mouse tau, and were immunoreactive with antibodies to 4R tau, phosphotau, misfolded tau, ubiquitin, and p62. Although local hyperphosphorylation of tau was increased in the dentate gyrus in THY-Tau22 mice, the development of neurofibrillary tangles made of mutant human tau was not accelerated in the hippocampus, indicating that wild-type human PHFs were inefficient in seeding tau aggregates made of G272V/P301S mutant human tau. Our results indicate thus that injection of human wild-type Alzheimer PHF seeded aggregation of wild-type murine tau into an argyrophilic 4R tau pathology, and constitutes an interesting model independent of expression of a mutant tau protein.info:eu-repo/semantics/publishe

Membrane binding, internalization, and sorting of alpha-synuclein in the cell

Abstract Alpha-synuclein (aSyn) plays a crucial role in Parkinson’s disease (PD) and other synucleinopathies, since it misfolds and accumulates in typical proteinaceous inclusions. While the function of aSyn is thought to be related to vesicle binding and trafficking, the precise molecular mechanisms linking aSyn with synucleinopathies are still obscure. aSyn can spread in a prion-like manner between interconnected neurons, contributing to the propagation of the pathology and to the progressive nature of synucleinopathies. Here, we investigated the interaction of aSyn with membranes and trafficking machinery pathways using cellular models of PD that are amenable to detailed molecular analyses. We found that different species of aSyn can enter cells and form high molecular weight species, and that membrane binding properties are important for the internalization of aSyn. Once internalized, aSyn accumulates in intracellular inclusions. Interestingly, we found that internalization is blocked in the presence of dynamin inhibitors (blocked membrane scission), suggesting the involvement of the endocytic pathway in the internalization of aSyn. By screening a pool of small Rab-GTPase proteins (Rabs) which regulate membrane trafficking, we found that internalized aSyn partially colocalized with Rab5A and Rab7. Initially, aSyn accumulated in Rab4A-labelled vesicles and, at later stages, it reached the autophagy-lysosomal pathway (ALP) where it gets degraded. In total, our study emphasizes the importance of membrane binding, not only as part of the normal function but also as an important step in the internalization and subsequent accumulation of aSyn. Importantly, we identified a fundamental role for Rab proteins in the modulation of aSyn processing, clearance and spreading, suggesting that targeting Rab proteins may hold important therapeutic value in PD and other synucleinopathies

Environmental and genetic factors support the dissociation between α-synuclein aggregation and toxicity

Synucleinopathies are a group of progressive disorders characterized by the abnormal aggregation and accumulation of α-synuclein (aSyn), an abundant neuronal protein that can adopt different conformations and biological properties. Recently, aSyn pathology was shown to spread between neurons in a prion-like manner. Proteins like aSyn that exhibit self-propagating capacity appear to be able to adopt different stable conformational states, known as protein strains, which can be modulated both by environmental and by protein-intrinsic factors. Here, we analyzed these factors and found that the unique combination of the neurodegeneration-related metal copper and the pathological H50Q aSyn mutation induces a significant alteration in the aggregation properties of aSyn. We compared the aggregation of WT and H50Q aSyn with and without copper, and assessed the effects of the resultant protein species when applied to primary neuronal cultures. The presence of copper induces the formation of structurally different and less-damaging aSyn aggregates. Interestingly, these aggregates exhibit a stronger capacity to induce aSyn inclusion formation in recipient cells, which demonstrates that the structural features of aSyn species determine their effect in neuronal cells and supports a lack of correlation between toxicity and inclusion formation. In total, our study provides strong support in favor of the hypothesis that protein aggregation is not a primary cause of cytotoxicity