α-Synuclein Fibril, Ribbon and Fibril-91 Amyloid Polymorphs Generation for Structural Studies.

The human α-synuclein protein, identified as one of the main markers of Parkinson's disease, is a 140-amino acid thermostable protein that can easily be overexpressed in E. coli. The purification protocol determines the ability of the protein to assemble into amyloid fibrils of well-defined structures. Here, we describe the purification and assembly protocols to obtain three well-characterized amyloid forms (ribbon, fibrils, and fibril-91) used to assess their activity in biochemical and cellular assays or to investigate their atomic structure by cryo-electron microscopy and solid-state NMR.

Dissecting aggregation and seeding dynamics of α-Syn polymorphs using the phasor approach to FLIM.

Synucleinopathies are a heterogenous group of neurodegenerative diseases characterized by the progressive accumulation of pathological α-synuclein (α-Syn). The importance of structural polymorphism of α-Syn assemblies for distinct synucleinopathies and their progression is increasingly recognized. However, the underlying mechanisms are poorly understood. Here we use fluorescence lifetime imaging microscopy (FLIM) to investigate seeded aggregation of α-Syn in a biosensor cell line. We show that conformationally distinct α-Syn polymorphs exhibit characteristic fluorescence lifetimes. FLIM further revealed that α-Syn polymorphs were differentially processed by cellular clearance pathways, yielding fibrillar species with increased seeding capacity. Thus, FLIM is not only a powerful tool to distinguish different amyloid structures, but also to monitor the dynamic process of amyloid remodeling by the cellular environment. Our data suggest that the accumulation of highly seeding competent degradation products for particular polymorphs may account for accelerated disease progression in some patients.

Human pericytes degrade diverse α-synuclein aggregates.

Parkinson's disease (PD) is a progressive, neurodegenerative disorder characterised by the abnormal accumulation of α-synuclein (α-syn) aggregates. Central to disease progression is the gradual spread of pathological α-syn. α-syn aggregation is closely linked to progressive neuron loss. As such, clearance of α-syn aggregates may slow the progression of PD and lead to less severe symptoms. Evidence is increasing that non-neuronal cells play a role in PD and other synucleinopathies such as Lewy body dementia and multiple system atrophy. Our previous work has shown that pericytes-vascular mural cells that regulate the blood-brain barrier-contain α-syn aggregates in human PD brains. Here, we demonstrate that pericytes efficiently internalise fibrillar α-syn irrespective of being in a monoculture or mixed neuronal cell culture. Pericytes cleave fibrillar α-syn aggregates (Fibrils, Ribbons, fibrils65, fibrils91 and fibrils110), with cleaved α-syn remaining present for up to 21 days. The number of α-syn aggregates/cell and average aggregate size depends on the type of strain, but differences disappear within 5 five hours of treatment. Our results highlight the role brain vasculature may play in reducing α-syn aggregate burden in PD.

Impact of α-Synuclein Fibrillar Strains and ß-Amyloid Assemblies on Mouse Cortical Neurons Endo-Lysosomal Logistics.

Endosomal transport and positioning cooperate in the establishment of neuronal compartment architecture, dynamics, and function, contributing to neuronal intracellular logistics. Furthermore, dysfunction of endo-lysosomal has been identified as a common mechanism in neurodegenerative diseases. Here, we analyzed endo-lysosomal transport when α-synuclein (α-syn) fibrillar polymorphs, ß-amyloid (Aß) fibrils, and oligomers were externally applied on primary cultures of mouse cortical neurons. To measure this transport, we used a simple readout based on the spontaneous endocytosis in cultured neurons of fluorescent nanodiamonds (FNDs), a perfectly stable nano-emitter, and the subsequent automatic extraction and quantification of their directed motions at high-throughput. α-Syn fibrillar polymorphs, Aß fibrils, and oligomers induce a 2-fold decrease of the fraction of nanodiamonds transported along microtubules, while only slightly reducing their interaction with cortical neurons. This important decrease in moving endosomes is expected to have a huge impact on neuronal homeostasis. We next assessed lysosomes dynamics, using LysoTracker. Neurons exposure to Aß oligomers led to an increase in the number of lysosomes, a decrease in the fraction of moving lysosome and an increase in their size, reminiscent of that found in APP transgenic model of Alzheimer's disease. We then analyzed the effect of α-syn fibrillar polymorphs, Aß fibrils, and oligomers on endosomal and lysosomal transport and quantified directed transport of those assemblies within cortical neurons. We report different impacts on endosomal and lysosomal transport parameters and differences in the trajectory lengths of cargoes loaded with pathogenic protein assemblies. Our results suggest that intraneuronal pathogenic protein aggregates internalization and transport may represent a target for novel neuroprotective therapeutic strategies.

Establishment of a human induced pluripotent stem cell neuronal model for identification of modulators of A53T α-synuclein levels and aggregation.

Inhibiting formation or promoting degradation of α-synuclein aggregates are among the therapeutical approaches under investigation as disease-modifying treatment strategies for Parkinson's disease. To support these developments, several in vitro models based on seeded α-synuclein aggregation have been established in immortalized cell lines and murine primary neurons. Here, we report on a humanized model with a reproducibility and throughput that enables its use in supporting target identification and validation in pharmacological research. A human induced pluripotent stem cell (iPSC) line was genetically modified to express HA-tagged α-synuclein with the point mutation in position 53 from Alanine to Threonine (A53T) under an inducible system and differentiated into cortical neurons expressing neuronal markers and exhibiting spontaneous activity. Intracellular α-synuclein aggregation was triggered by exposure to exogenous added fibrillated recombinant wild-type human α-synuclein fibrils91 and demonstrated by several endpoints; the formation of Triton-insoluble SDS-soluble α-synuclein, biochemically in a fluorescence resonance energy transfer based aggregation assay and by immunocytochemistry of phosphorylated α-synuclein positive puncta. We demonstrate the feasibility of upscaling the iPSC neuron production for drug discovery and that the model has a suitable dynamic range allowing for both detection of increased and decreased α-synuclein aggregation. Moreover, gene modulation is feasible using siRNAs, making the model suitable for genetic screening for modulators of α-synuclein aggregation. Data on effects of USP8, USP13 and USP9X knockdown on α-synuclein expression and aggregation contradicts published data from immortalized cell lines and murine systems. This highlight the importance of including humanized neuronal models in the confirmation of biological mechanisms in specific variations of Parkinson's disease.

Microglial inclusions and neurofilament light chain release follow neuronal α-synuclein lesions in long-term brain slice cultures.

BACKGROUND: Proteopathic brain lesions are a hallmark of many age-related neurodegenerative diseases including synucleinopathies and develop at least a decade before the onset of clinical symptoms. Thus, understanding of the initiation and propagation of such lesions is key for developing therapeutics to delay or halt disease progression. METHODS: Alpha-synuclein (αS) inclusions were induced in long-term murine and human slice cultures by seeded aggregation. An αS seed-recognizing human antibody was tested for blocking seeding and/or spreading of the αS lesions. Release of neurofilament light chain (NfL) into the culture medium was assessed. RESULTS: To study initial stages of α-synucleinopathies, we induced αS inclusions in murine hippocampal slice cultures by seeded aggregation. Induction of αS inclusions in neurons was apparent as early as 1week post-seeding, followed by the occurrence of microglial inclusions in vicinity of the neuronal lesions at 2-3 weeks. The amount of αS inclusions was dependent on the type of αS seed and on the culture's genetic background (wildtype vs A53T-αS genotype). Formation of αS inclusions could be monitored by neurofilament light chain protein release into the culture medium, a fluid biomarker of neurodegeneration commonly used in clinical settings. Local microinjection of αS seeds resulted in spreading of αS inclusions to neuronally connected hippocampal subregions, and seeding and spreading could be inhibited by an αS seed-recognizing human antibody. We then applied parameters of the murine cultures to surgical resection-derived adult human long-term neocortical slice cultures from 22 to 61-year-old donors. Similarly, in these human slice cultures, proof-of-principle induction of αS lesions was achieved at 1week post-seeding in combination with viral A53T-αS expressions. CONCLUSION: The successful translation of these brain cultures from mouse to human with the first reported induction of human αS lesions in a true adult human brain environment underlines the potential of this model to study proteopathic lesions in intact mouse and now even aged human brain environments.

Multiple Site-Specific Phosphorylation of IDPs Monitored by NMR.

In line with their high accessibility, disordered proteins are exquisite targets of kinases. Eukaryotic organisms use the so-called intrinsically disordered proteins (IDPs) or intrinsically disordered regions of proteins (IDRs) as molecular switches carrying intracellular information tuned by reversible phosphorylation schemes. Solvent-exposed serines and threonines are abundant in IDPs, and, consistently, kinases often modify disordered regions of proteins at multiple sites. In this context, nuclear magnetic resonance (NMR) spectroscopy provides quantitative, residue-specific information that permits mapping of phosphosites and monitoring of their individual kinetics. Hence, NMR monitoring emerges as an in vitro approach, complementary to mass-spectrometry or immuno-blotting, to characterize IDP phosphorylation comprehensively. Here, we describe in detail generic protocols for carrying out NMR monitoring of IDP phosphorylation, and we provide a number of practical insights that improve handiness and reproducibility of this method.

Sensitivity-Enhanced 13 C-NMR Spectroscopy for Monitoring Multisite Phosphorylation at Physiological Temperature and pH.

Abundant phosphorylation events control the activity of nuclear proteins involved in gene regulation and DNA repair. These occur mostly on disordered regions of proteins, which often contain multiple phosphosites. Comprehensive and quantitative monitoring of phosphorylation reactions is theoretically achievable at a residue-specific level using 1 H-15 N NMR spectroscopy, but is often limited by low signal-to-noise at pH>7 and T>293 K. We have developed an improved 13 Cα-13 CO correlation NMR experiment that works equally at any pH or temperature, that is, also under conditions at which kinases are active. This allows us to obtain atomic-resolution information in physiological conditions down to 25 µm. We demonstrate the potential of this approach by monitoring phosphorylation reactions, in the presence of purified kinases or in cell extracts, on a range of previously problematic targets, namely Mdm2, BRCA2, and Oct4.