13C and 15N Resonance Assignments of Alpha Synuclein Fibrils Amplified from Lewy Body Dementia Tissue

Fibrils of the protein α-synuclein (Asyn) are implicated in the pathogenesis of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. Numerous forms of Asyn fibrils have been studied by solid-state NMR and resonance assignments have been reported. Here, we report a new set of 13C, 15N assignments that are unique to fibrils obtained by amplification from postmortem brain tissue of a patient diagnosed with Lewy Body Dementia.

. Growing evidence indicates that distinct polymorphs are associated with pathologic Asyn accumulation in α-synucleinopathies, as detailed by previously reported in vivo studies (Frieg et al. 2022, Schweighauser et al. 2020, Yang et al. 2022). Therefore, structural determination of these brils is vital for advancing the understanding of disease etiology, and to aid the development of polymorph-speci c clinical diagnostic tools and novel therapeutics.
We isolated insoluble Asyn brils from postmortem LBD tissue. Then, to analyze LBD bril structure by SSNMR, we ampli ed the Asyn bril seeds using uniform [ 13 C, 15 N] labeled wild-type Asyn. Here, we report the 13 C and 15 N chemical shifts for the ampli ed Asyn brils from an LBD autopsy case.. The spectra of these ex vivo brils exhibit resonances that differ from those of previously reported in vitro bril preparations. These ndings demonstrate a different arrangement of β-strands, supporting the hypothesis that bril structure is directly linked to disease phenotype.

Methods And Experiments
Protein expression and puri cation Expression of uniform [ 13 C, 15 15 N]Bioexpress (Cambridge Isotope Laboratories, Inc., Tewksbury, MA), 1 mL/L BME vitamins (Sigma), and 90 μg/mL kanamycin. After a preliminary growth in medium containing natural abundance (NA) isotopes, the cells were transferred to the labeling medium at 37 ºC to an OD 600 of 1.2, at which point the temperature was reduced to 25 ºC and protein expression induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown for 15 h to a nal OD 600 of 4.1 and harvested.
Protein puri cation was done as described previously (Barclay et al. 2018). Brie y, cells were lysed chemically in the presence of Turbonuclease (Sigma) to digest nucleic acids. Puri cation began with a heat denaturation of the cleared lysate, followed by ammonium sulfate precipitation (Kloepper et al. 2006). The resolubilized protein was bound to QFF anion exchange resin (GE Healthcare Life Sciences, Marlborough, MA) and eluted using a linear gradient of 0.2-0.6 M NaCl. Fractions containing Asyn monomer, which eluted at about 0.3 M NaCl, were pooled, concentrated, and run over a 26/60 Sephacryl S-200 HR gel ltration column (GE Healthcare Life Sciences) equilibrated in 50 mM Tris-HCl, 100 mM NaCl, pH 8 buffer. Fractions were pooled, concentrated to ~20 mg/mL Asyn, and dialyzed at 4 ºC into 10 mM Tris-HCl pH 7.6, 50 mM NaCl, 1 mM DTT, and stored at a concentration of ~14 mg/mL at -80 ºC until use. Yields were 95 mg puri ed AS protein/L growth medium for the uniform [ 13 C, 15 N] labeled monomer.
Preparation of Insoluble fraction seeds from LBD, MSA and control postmortem tissue The protocol to sequentially extract human postmortem brain tissue was adapted from Appel-Cresswell et al 2 .
Brie y, gray matter dissected from tissue was sequentially homogenized in four buffers (3 ml/g wet weight of tissue) using Kimble Chase Konte TM dounce tissue grinders (KT885300-0002). In the rst step, 300mg of dissected grey matter tissue was homogenized using 20 strokes of Pestle A in High Salt (HS) buffer (50 mM Tris-HCl pH 7.5, 750 mM NaCl, 5 mM EDTA plus Sigma P2714 Protease Inhibitor (PI) cocktail). The homogenate was centrifuged at 100,000 ×g for 20 min at 4 °C and the pellet was homogenized in the next buffer using 20 strokes of Pestle B. Extractions using Pestle B were performed in HS buffer with 1% Triton X-100 with PI, then HS buffer with 1% Triton X-100 and 1 M sucrose, and with 50 mM Tris-HCl, pH 7.4 buffer. In the nal centrifugation, the resulting pellet was resuspended in 50 mM Tris-HCl, pH 7.4 buffer (3 ml/g wet weight of tissue). The aliquots of insoluble fraction were stored at -80 °C until use. Similar extraction protocol was followed for LBD, MSA and control cases.

Ampli cation of isotopically labelled LBD brils from LBD insoluble fraction seeds
We ampli ed LBD-brils from gray matter dissected from the caudate region. We incubated insoluble fraction seeds with an Asyn monomer preparation containing isotopically labeled Asyn monomer supplemented with control fraction. The control fraction preparation was derived from E.Coli transformed with an empty expression vector, and was puri ed with the same protocol as the natural abundance Asyn monomer (JBC, 2017 V292, Pg9034). Asyn monomer and control fraction was ltered through a 50k MWCO Amicon Ultra centrifugation lter (Millipore, UFC805204) before use, to remove any preformed aggregates.
Insoluble fraction (10 µL) containing 3.3 µg wet wt. of tissue was bought to a nal volume of 30 µL by addition of 20 mM Tris-HCl, pH 8.0 plus 100 mM NaCl buffer ( bril buffer) in a 1.7mL microcentrifuge tube. The insoluble fraction was sonicated for 2 min at amplitude 50 in a bath sonicator (Qsonica model Q700) with a cup horn (5.5 inch) attachment at 4 °C. To the sonicated seeds, 1.5 µL of 2 % Triton X-100 was added. To this mixture, 50k Amicon ultra ltered isotopically labelled Asyn monomer was added to a nal concentration of 2 mg/mL in a nal volume of 100 µL. This mixture underwent quiescent incubation at 37 °C for 3 days, completing the 1 st round of sonication plus incubation. After the rst round, the mixture was sonicated at 1 min at amplitude 50, and then an additional 300 µL of 2 mg/mL Asyn monomer was added. The mixture underwent quiescent incubation at 37 °C for 2 days (2 nd round). Then, sonication for 1 min at amplitude 50 and quiescent incubation for 2 days was repeated for the third round, followed by sonication for 1 min at amplitude 50 and quiescent incubation for 3 days for the 4 th round. At the end of 4 th round, LBD-ampli ed brils were stored at 4°C until use.
Further expansion of the LBD-ampli ed brils was performed by centrifuging 60 μL of 4 th round LBD-ampli ed brils at 21,000 xg for 15 min at 4 °C. The pellet was resuspended in 100 µL of bril buffer and sonicated for 1 min at ampli ed 50. To this mixture, Asyn monomer was added to a nal concentration of 2 mg/mL in a nal volume of 400 µL in bril buffer. This mix was quiescently incubated at 37 °C for 2 days (5 th round). At the end of 5 th round, samples were centrifuged at 21,000 xg for 15 min at 4 °C and the top 300 µL of spent Asyn monomer was moved to a separate tube. The pellet was resuspended by trituration and sonicated for 1 min at ampli ed 50. After sonication, the previously removed 300 µL of 5 th round monomer was added back. Next, an additional 2.5 mg/mL of Asyn monomer was added to bring the total volume to 800 µL. This mixture was incubated at 37 °C for 2 days to complete 6 th round of incubation. The increased monomer concentration (2.5 mg/mL instead of 2 mg/mL) was calculated based on the average decrease in free Asyn monomer due to its incorporation into ampli ed brils. The 6 th round brils were stored at 4 °C until use.

Assignments And Data Deposition Chemical Shift Assignments and Extent of Assignments
Chemical shift assignments were performed for the LBD Asyn brils ampli ed using uniform [ 13 C, 15 N] labeling (uCN). Resonance assignments were determined using 2D 13 C-13 C, 2D 15 N-13 Cα, 2D 15 N-13 C', 3D 15 N-13 Cα-13 CX, 3D 15 N-13 C'-13 CX, 3D 15 N-13 C'- 13 Cα and 3D 13 Cα-15 N-13 C' data sets following standard procedures (Comellas and Rienstra, 2013;Higman, 2018). The complete list of data sets used for performing assignments using 13 Cdetection is presented in Supplementary Table 1. The 2D 13 C-13 C spectrum serves as a conformational ngerprint of the bril and provides some initial insights into the structure Figure 1A. Globally, the resolved peaks display line widths, including scalar couplings, of < 0.4 ppm in the direct 13 C dimension, similar to those observed for in vitro Asyn brils and indicative of a highly ordered core (Barclay et al., 2018;Comellas et al., 2011a). Particular residue types including Thr, Val, and Gly, as well as select spin systems including L38, N65, Q79, I88, and F94 are particularly well resolved in the 2D. The Lys and Glu regions, which account for 20% of the primary sequence between residues 30 and 100, are broad and poorly resolved as with the in vitro form, indicating disorder or partial mobility for the majority of these residues. In contrast to the in vitro form, the Ala regions are poorly resolved in the 2D 13 C-13 C, but the signals are of comparable intensity to the resolved regions, indicating that the alanines are highly ordered but of similar structure throughout the core. Interestingly, S87 displays two sets of peaks that combine to approximately half of the intensity of the strong peaks, while I88 shows broadening for the peaks arising from backbone correlations, suggesting at least two conformations for this region. In the 3D spectra, a few additional residues, namely A76 and V77, also show two sets of peaks but for the side chain atoms. In addition, the Asyn sequence contains 10 Thr residues all located between residues 22 and 92, but at least 11 appear in the 2D 13 C-13 C spectrum. The 2D 15 N-13 C' ( Figure 1A) and 15 N-13 Cα ( Figure 1B) spectra also serve as structural ngerprints for the bril, with a focus on the backbone atoms.
Critically, these highlight the bene t of adding a 15 N dimension to disambiguate shifts, particularly for the Ala and Gly regions, which comprise 30% of the primary sequence between residues 30 and 100, as well as for key core residues like V71, V74, T75 and V77. Overall, there appears to be a predominant de ned conformation that likely displays some localized heterogeneity. Figure 2 demonstrates representative strips corresponding to assignments from 3D 15 N-13 Cα-13 CX, 3D 15 N-13 C'-13 CX, and 3D 13 Cα-15 N-13 C' from T72 to A76.     Example backbone assignment strip from T72 to A76 demonstrating connectivity and sidechain assignments from a 3D 15 N-13 Cα-13 CX correlation (black), a 3D 15 N-13 C'-13 CX correlation(red), and a 3D 13 Cα-15 N-13 C'correlation (blue).