A Targetable N-Terminal Motif Orchestrates α-Synuclein Oligomer-to-Fibril Conversion.

Oligomeric species populated during α-synuclein aggregation are considered key drivers of neurodegeneration in Parkinson's disease. However, the development of oligomer-targeting therapeutics is constrained by our limited knowledge of their structure and the molecular determinants driving their conversion to fibrils. Phenol-soluble modulin α3 (PSMα3) is a nanomolar peptide binder of α-synuclein oligomers that inhibits aggregation by blocking oligomer-to-fibril conversion. Here, we investigate the binding of PSMα3 to α-synuclein oligomers to discover the mechanistic basis of this protective activity. We find that PSMα3 selectively targets an α-synuclein N-terminal motif (residues 36-61) that populates a distinct conformation in the mono- and oligomeric states. This α-synuclein region plays a pivotal role in oligomer-to-fibril conversion as its absence renders the central NAC domain insufficient to prompt this structural transition. The hereditary mutation G51D, associated with early onset Parkinson's disease, causes a conformational fluctuation in this region, leading to delayed oligomer-to-fibril conversion and an accumulation of oligomers that are resistant to remodeling by molecular chaperones. Overall, our findings unveil a new targetable region in α-synuclein oligomers, advance our comprehension of oligomer-to-amyloid fibril conversion, and reveal a new facet of α-synuclein pathogenic mutations.

Efficient 18.8 T MAS-DNP NMR reveals hidden side chains in amyloid fibrils.

Amyloid fibrils are large and insoluble protein assemblies composed of a rigid core associated with a cross-ß arrangement rich in ß-sheet structural elements. It has been widely observed in solid-state NMR experiments that semi-rigid protein segments or side chains do not yield easily observable NMR signals at room temperature. The reasons for the missing peaks may be due to the presence of unfavorable dynamics that interfere with NMR experiments, which result in very weak or unobservable NMR signals. Therefore, for amyloid fibrils, semi-rigid and dynamically disordered segments flanking the amyloid core are very challenging to study. Here, we show that high-field dynamic nuclear polarization (DNP), an NMR hyperpolarization technique typically performed at low temperatures, can circumvent this issue because (i) the low-temperature environment (~ 100 K) slows down the protein dynamics to escape unfavorable detection regime, (ii) DNP improves the overall NMR sensitivity including those of flexible side chains, and (iii) efficient cross-effect DNP biradicals (SNAPol-1) optimized for high-field DNP (≥ 18.8 T) are employed to offer high sensitivity and resolution suitable for biomolecular NMR applications. By combining these factors, we have successfully established an impressive enhancement factor of ε ~ 50 on amyloid fibrils using an 18.8 T/ 800 MHz magnet. We have compared the DNP efficiencies of M-TinyPol, NATriPol-3, and SNAPol-1 biradicals on amyloid fibrils. We found that SNAPol-1 (with ε ~ 50) outperformed the other two radicals. The MAS DNP experiments revealed signals of flexible side chains previously inaccessible at conventional room-temperature experiments. These results demonstrate the potential of MAS-DNP NMR as a valuable tool for structural investigations of amyloid fibrils, particularly for side chains and dynamically disordered segments otherwise hidden at room temperature.

Cryo-EM observation of the amyloid key structure of polymorphic TDP-43 amyloid fibrils.

The transactive response DNA-binding protein-43 (TDP-43) is a multi-facet protein involved in phase separation, RNA-binding, and alternative splicing. In the context of neurodegenerative diseases, abnormal aggregation of TDP-43 has been linked to amyotrophic lateral sclerosis and frontotemporal lobar degeneration through the aggregation of its C-terminal domain. Here, we report a cryo-electron microscopy (cryo-EM)-based structural characterization of TDP-43 fibrils obtained from the full-length protein. We find that the fibrils are polymorphic and contain three different amyloid structures. The structures differ in the number and relative orientation of the protofilaments, although they share a similar fold containing an amyloid key motif. The observed fibril structures differ from previously described conformations of TDP-43 fibrils and help to better understand the structural landscape of the amyloid fibril structures derived from this protein.

Neurons with Cat's Eyes: A Synthetic Strain of α-Synuclein Fibrils Seeding Neuronal Intranuclear Inclusions.

The distinct neuropathological features of the different α-Synucleinopathies, as well as the diversity of the α-Synuclein (α-Syn) intracellular inclusion bodies observed in post mortem brain sections, are thought to reflect the strain diversity characterizing invasive α-Syn amyloids. However, this "one strain, one disease" view is still hypothetical, and to date, a possible disease-specific contribution of non-amyloid factors has not been ruled out. In Multiple System Atrophy (MSA), the buildup of α-Syn inclusions in oligodendrocytes seems to result from the terminal storage of α-Syn amyloid aggregates first pre-assembled in neurons. This assembly occurs at the level of neuronal cytoplasmic inclusions, and even earlier, within neuronal intranuclear inclusions (NIIs). Intriguingly, α-Syn NIIs are never observed in α-Synucleinopathies other than MSA, suggesting that these inclusions originate (i) from the unique molecular properties of the α-Syn fibril strains encountered in this disease, or alternatively, (ii) from other factors specifically dysregulated in MSA and driving the intranuclear fibrillization of α-Syn. We report the isolation and structural characterization of a synthetic human α-Syn fibril strain uniquely capable of seeding α-Syn fibrillization inside the nuclear compartment. In primary mouse cortical neurons, this strain provokes the buildup of NIIs with a remarkable morphology reminiscent of cat's eye marbles (see video abstract). These α-Syn inclusions form giant patterns made of one, two, or three lentiform beams that span the whole intranuclear volume, pushing apart the chromatin. The input fibrils are no longer detectable inside the NIIs, where they become dominated by the aggregation of endogenous α-Syn. In addition to its phosphorylation at S129, α-Syn forming the NIIs acquires an epitope antibody reactivity profile that indicates its organization into fibrils, and is associated with the classical markers of α-Syn pathology p62 and ubiquitin. NIIs are also observed in vivo after intracerebral injection of the fibril strain in mice. Our data thus show that the ability to seed NIIs is a strain property that is integrally encoded in the fibril supramolecular architecture. Upstream alterations of cellular mechanisms are not required. In contrast to the lentiform TDP-43 NIIs, which are observed in certain frontotemporal dementias and which are conditional upon GRN or VCP mutations, our data support the hypothesis that the presence of α-Syn NIIs in MSA is instead purely amyloid-strain-dependent.

Examining the Transient Dark State in Protein-Quantum Dot Interaction by Relaxation-Based Solution NMR.

We probed the "dark" state involved in the protein-quantum dot (QD) interaction using a relaxation-based solution nuclear magnetic resonance (NMR) approach. We examined the dynamics and exchange kinetics of the ubiquitin-CdTe model system, which undergoes a fast exchange in the transverse relaxation time scale. We applied the recently developed dark-state exchange saturation transfer (DEST), lifetime line broadening (ΔR2), and exchange-induced chemical shift (δex) solution NMR techniques to obtain a residue-specific binding behavior of the protein on the QD surface. The variation in the estimated 15N-R2bound values clearly shows the dynamic nature of bound Ub. Upon mapping the amino acid residues showing a faster relaxation rate on the electrostatic potential surface of the protein, we have determined that the interaction is preferably electrostatic, and the amino acid residues involved in binding lie on the positively charged surface of the protein. We believe that our experimental approach should provide more in-depth knowledge to engineer new hybrid protein-QD systems in the future.