Host oligodendrogliopathy and α-synuclein strains dictate disease severity in multiple system atrophy.

Multiple system atrophy is a progressive neurodegenerative disease with prominent autonomic and motor features. During early stages, different subtypes of the disease are distinguished by their predominant parkinsonian or cerebellar symptoms, reflecting its heterogeneous nature. The pathognomonic feature of multiple system atrophy is the presence of α-synuclein (αSyn) protein deposits in oligodendroglial cells. αSyn can assemble in specific cellular or disease environments and form αSyn strains with unique structural features, but the ability of αSyn strains to propagate in oligodendrocytes remains elusive. Recently, it was shown that αSyn strains with related conformations exist in the brains of patients. Here, we investigated whether different αSyn strains can influence multiple system atrophy progression in a strain-dependent manner. To this aim, we injected two recombinant αSyn strains (fibrils and ribbons) in multiple system atrophy transgenic mice and found that they determined disease severity in multiple system atrophy via host-restricted and cell-specific pathology in vivo. αSyn strains significantly impact disease progression in a strain-dependent way via oligodendroglial, neurotoxic and immune-related mechanisms. Neurodegeneration and brain atrophy were accompanied by unique microglial and astroglial responses and the recruitment of central and peripheral immune cells. The differential activation of microglial cells correlated with the structural features of αSyn strains both in vitro and in vivo. Spectral analysis showed that ribbons propagated oligodendroglial inclusions that were structurally distinct from those of fibrils, with resemblance to oligodendroglial inclusions, in the brains of patients with multiple system atrophy. This study, therefore, shows that the multiple system atrophy phenotype is governed by both the nature of the αSyn strain and the host environment and that by injecting αSyn strains into an animal model of the disease, a more comprehensive phenotype can be established.

Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes.

Extracellular vesicles (EVs) have emerged as key players in cell-to-cell communication in both physiological and pathological processes in the Central Nervous System. Thus far, the intracellular pathways involved in uptake and trafficking of EVs within different cell types of the brain are poorly understood. In our study, the endocytic processes and subcellular sorting of EVs were investigated in primary glial cells, particularly linked with the EV-associated α-synuclein (α-syn) transmission. Mouse microglia and astrocytic primary cultures were incubated with DiI-stained mouse brain-derived EVs. The internalization and trafficking pathways were analyzed in cells treated with pharmacological reagents that block the major endocytic pathways. Brain-derived EVs were internalized by both glial cell types; however, uptake was more efficient in microglia than in astrocytes. Colocalization of EVs with early and late endocytic markers (Rab5, Lamp1) indicated that EVs are sorted to endo-lysosomes for subsequent processing. Blocking actin-dependent phagocytosis and/or macropinocytosis with Cytochalasin D or EIPA inhibited EV entry into glial cells, whereas treatment with inhibitors that strip cholesterol off the plasma membrane, induced uptake, however differentially altered endosomal sorting. EV-associated fibrillar α-Syn was efficiently internalized and detected in Rab5- and Lamp1-positive compartments within microglia. Our study strongly suggests that EVs enter glial cells through phagocytosis and/or macropinocytosis and are sorted to endo-lysosomes for subsequent processing. Further, brain-derived EVs serve as scavengers and mediate cell-to-glia transfer of pathological α-Syn which is also targeted to the endolysosomal pathway, suggesting a beneficial role in microglia-mediated clearance of toxic protein aggregates, present in numerous neurodegenerative diseases.

Clustering of Tau fibrils impairs the synaptic composition of α3-Na+/K+-ATPase and AMPA receptors.

Tau assemblies have prion-like properties: they propagate from one neuron to another and amplify by seeding the aggregation of endogenous Tau. Although key in prion-like propagation, the binding of exogenous Tau assemblies to the plasma membrane of naïve neurons is not understood. We report that fibrillar Tau forms clusters at the plasma membrane following lateral diffusion. We found that the fibrils interact with the Na+/K+-ATPase (NKA) and AMPA receptors. The consequence of the clustering is a reduction in the amount of α3-NKA and an increase in the amount of GluA2-AMPA receptor at synapses. Furthermore, fibrillar Tau destabilizes functional NKA complexes. Tau and α-synuclein aggregates often co-exist in patients' brains. We now show evidences for cross-talk between these pathogenic aggregates with α-synuclein fibrils dramatically enhancing fibrillar Tau clustering and synaptic localization. Our results suggest that fibrillar α-synuclein and Tau cross-talk at the plasma membrane imbalance neuronal homeostasis.

Dipeptide repeat derived from C9orf72 hexanucleotide expansions forms amyloids or natively unfolded structures in vitro.

The abnormal repetition of the hexanucleotide GGGGCC within the C9orf72 gene is the most common genetic cause of both Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). Different hypothesis have been proposed to explain the pathogenicity of this mutation. Among them, the production of aberrant proteins called Dipeptide Repeat Proteins (DPR) from the repeated sequence. Those proteins are of interest, as they are toxic and form insoluble deposits in patient brains. In this study, we characterized the structural features of three different DPR encoded by the hexanucleotide repeat GGGGCC, namely poly-GA, poly-GP and poly-PA. We showed that DPR are natively unstructured proteins and that only poly-GA forms in vitro fibrillary aggregates. Poly-GA fibrils are of amyloid nature as revealed by their high content in beta sheets. They neither bind Thioflavin T nor Primuline, the commonly used amyloid fluorescent dyes. Remarkably, not all of the poly-GA primary structure was part of fibrils amyloid core.

C9ORF72-derived poly-GA DPRs undergo endocytic uptake in iAstrocytes and spread to motor neurons.

Dipeptide repeat (DPR) proteins are aggregation-prone polypeptides encoded by the pathogenic GGGGCC repeat expansion in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. In this study, we focus on the role of poly-GA DPRs in disease spread. We demonstrate that recombinant poly-GA oligomers can directly convert into solid-like aggregates and form characteristic ß-sheet fibrils in vitro. To dissect the process of cell-to-cell DPR transmission, we closely follow the fate of poly-GA DPRs in either their oligomeric or fibrillized form after administration in the cell culture medium. We observe that poly-GA DPRs are taken up via dynamin-dependent and -independent endocytosis, eventually converging at the lysosomal compartment and leading to axonal swellings in neurons. We then use a co-culture system to demonstrate astrocyte-to-motor neuron DPR propagation, showing that astrocytes may internalise and release aberrant peptides in disease pathogenesis. Overall, our results shed light on the mechanisms of poly-GA cellular uptake and propagation, suggesting lysosomal impairment as a possible feature underlying the cellular pathogenicity of these DPR species.

Absence of Uptake and Prion-Like Spreading of Alpha-Synuclein and Tau After Intravitreal Injection of Preformed Fibrils.

Although very different in etiology and symptoms, numerous neurodegenerative diseases can be classified as proteinopathies. More so, evidence indicates that the key misfolded proteins at the basis of different neuropathies might share common mechanisms of propagation. As such, the prion-like spreading of protein aggregates through the neural network is subject of intensive research focus and requires adequate models. Here, we made use of the well-defined architecture and large accessibility of the visual system, of which the retinotopic connections represent a simple route of anterograde signaling and an elegant model to investigate transsynaptic, prion-like spreading. In two independent studies, uptake and seeding of alpha-synuclein and tau were examined after intravitreal injection of preformed fibrils. However, extracellular matrix components in the vitreous space and at the vitreoretinal surface appeared to act as a barrier for the entry of both fibrils into the retina. These results show that further experimental refinement is needed to fully realize the potential of the visual system as a model for studying the molecular and cellular mechanisms of anterograde, transsynaptic spreading of prion-like proteins.

CX3CL1 homo-oligomerization drives cell-to-cell adherence.

During inflammatory response, blood leukocytes adhere to the endothelium. This process involves numerous adhesion molecules, including a transmembrane chemokine, CX3CL1, which behaves as a molecular cluster. How this cluster assembles and whether this association has a functional role remain unknown. The analysis of CX3CL1 clusters using native electrophoresis and single molecule fluorescence kinetics shows that CX3CL1 is a homo-oligomer of 3 to 7 monomers. Fluorescence recovery after photobleaching assays reveal that the CX3CL1-transmembrane domain peptide self-associates in both cellular and acellular lipid environments, while its random counterpart (i.e. peptide with the same residues in a different order) does not. This strongly indicates that CX3CL1 oligomerization is driven by its intrinsic properties. According to the molecular modeling, CX3CL1 does not associate in compact bundles but rather with monomers linearly assembled side by side. Finally, the CX3CL1 transmembrane peptide inhibits both the CX3CL1 oligomerization and the adhesive function, while its random counterpart does not. This demonstrates that CX3CL1 oligomerization is mandatory for its adhesive potency. Our results provide a new direction to control CX3CL1-dependent cellular adherence in key immune processes.

Assessment of the efficacy of different procedures that remove and disassemble alpha-synuclein, tau and A-beta fibrils from laboratory material and surfaces.

α-synuclein fibrillar polymorphs, Tau and Aß 1-42 fibrillar assemblies have been shown to propagate, amplify and trigger the formation of protein deposits reminiscent of those present within the central nervous system of patients developing synucleinopathies, tauopathies and amyloid plaques after injection intracerebrally, intramuscularly, intraperitoneally or within the blood stream of model animals. They are thus hazardous and there is need for decontamination and inactivation procedures for laboratory surfaces and non-disposable material. We assessed the effectiveness of different reagents to clean and disassemble potentially pathogenic assemblies adsorbed on non-disposable materials in laboratories. We show that commercial detergents and SDS are way more suited to detach α-synuclein fibrillar polymorphs, Tau and Aß 1-42 fibrillar assemblies from contaminated surfaces and disassemble the fibrils than methods designed to decrease PrP prion infectivity. Our observations reveal that the choice of the most adapted cleaning procedure for one given protein assembly or fibrillar polymorph should integrate detergent's cleaning efficiency, material compatibility and capacity to dismantle assemblies. We provide an integrated representation where desorption and neutralization efficacy and surface compatibility are combined to facilitate the choice of the most adapted decontamination procedure. This representation, together with good laboratory practices, contributes to reducing potential health hazards associated to manipulating protein assemblies with prion-like properties.

Tunneling nanotube (TNT)-mediated neuron-to neuron transfer of pathological Tau protein assemblies.

A given cell makes exchanges with its neighbors through a variety of means ranging from diffusible factors to vesicles. Cells use also tunneling nanotubes (TNTs), filamentous-actin-containing membranous structures that bridge and connect cells. First described in immune cells, TNTs facilitate HIV-1 transfer and are found in various cell types, including neurons. We show that the microtubule-associated protein Tau, a key player in Alzheimer's disease, is a bona fide constituent of TNTs. This is important because Tau appears beside filamentous actin and myosin 10 as a specific marker of these fine protrusions of membranes and cytosol that are difficult to visualize. Furthermore, we observed that exogenous Tau species increase the number of TNTs established between primary neurons, thereby facilitating the intercellular transfer of Tau fibrils. In conclusion, Tau may contribute to the formation and function of the highly dynamic TNTs that may be involved in the prion-like propagation of Tau assemblies.