Inflammation promotes synucleinopathy propagation

www.nature.com/emm ARTICLE OPEN Inflammation promotes synucleinopathy propagation Tae-Kyung Kim1,2,14, Eun-Jin Bae 1,3,14, Byung Chul Jung1,11,14, Minsun Choi1, Soo Jean Shin1, Sung Jun Park1, Jeong Tae Kim1, ✉ Min Kyo Jung4, Ayse Ulusoy5, Mi-Young Song6,12, Jun Sung Lee1,13, He-Jin Lee7,8, Donato A. Di Monte5 and Seung-Jae Lee1,3,9,10 © The Author(s) 2022 1234567890();,: The clinical progression of neurodegenerative diseases correlates with the spread of proteinopathy in the brain. The current understanding of the mechanism of proteinopathy spread is far from complete. Here, we propose that inflammation is fundamental to proteinopathy spread. A sequence variant of α-synuclein (V40G) was much less capable of fibril formation than wild-type αsynuclein (WT-syn) and, when mixed with WT-syn, interfered with its fibrillation. However, when V40G was injected intracerebrally into mice, it induced aggregate spreading even more effectively than WT-syn. Aggregate spreading was preceded by sustained microgliosis and inflammatory responses, which were more robust with V40G than with WT-syn. Oral administration of an antiinflammatory agent suppressed aggregate spreading, inflammation, and behavioral deficits in mice. Furthermore, exposure of cells to inflammatory cytokines increased the cell-to-cell propagation of α-synuclein. These results suggest that the inflammatory microenvironment is the major driver of the spread of synucleinopathy in the brain. Experimental & Molecular Medicine (2022) 54:2148–2161; https://doi.org/10.1038/s12276-022-00895-w INTRODUCTION Protein aggregation is the major pathological hallmark of several neurodegenerative diseases, with different types of aggregates and distribution patterns characterizing each disease. Large-scale pathological postmortem examinations have indicated that protein aggregates in Alzheimer’s and Parkinson’s diseases spread from a few initial sites and progressively involve an increasing number of brain regions in a highly specific topographic sequence1,2. This spreading of pathological aggregates most likely occurs via direct cell-to-cell transfer of aggregation-prone pathogenic proteins, such as tau and α-synuclein3–6. Although the mechanism remains unclear, the aggregate spreading phenomenon has been verified in several model systems. The most commonly used animal model for studying aggregate spreading is mice injected intracerebrally with preformed fibrils (PFFs). A single injection of PFF causes aggregation of the corresponding protein (e.g., tau or α-synuclein) in different brain regions7. Features underlying the transmission of prion proteins have led researchers to presume that aggregate spread in Alzheimer’s disease and Parkinson’s disease also occurs by a mechanism known as templated conformational seeding8. However, this theory is apparently inconsistent with some experimental observations. For example, PFFs are rapidly cleared after injection, and there is always an incubation time before aggregates reappear7. Furthermore, injection of an α-synuclein variant lacking the critical region for fibrillation still resulted in α-synuclein aggregate spreading9–11. Thus, mechanisms other than templated seeding may contribute to the spread of protein aggregates. Here, we directly tested whether templated seeding is sufficient to explain protein aggregate spreading by using a specific sequence variant of α-synuclein (V40G) that cannot seed aggregation. MATERIALS AND METHODS Mutagenesis A mutation (V40G) was introduced into human wild-type α-synuclein (α-synuclein/pDual GC; Agilent Technologies, Santa Clara, CA, USA, #214503) using a QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, #200521). The primers used in this study are listed in Supplementary Table 5. Protein purification and fibril preparation WT-syn and V40G were expressed and purified as previously described12. Fibrillation was performed as previously described12. Human or mouse αsynuclein (200 μM in PBS) was incubated at 37 °C for 9 days with constant shaking at 1,050 rpm in the presence or absence of seeds. Circular dichroism (CD) spectroscopy CD analysis was performed as previously described12. All spectra were obtained by averaging 10 separate measurements. Transmission electron microscopy (TEM) TEM was performed as previously described12. The aged WT-syn and V40G were observed using a JEM1010 transmission electron microscope (JEOL, Akishima, Tokyo, Japan). 1 Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, Korea. 2Department of Exercise Physiology and Sport Science Institute, Korea National Sport University, Seoul 05541, Korea. 3Neuroscience Research Institute, Seoul National University College of Medicine, Seoul, South Korea. 4Neural Circuits Research Group, Korea Brain Research Institute, Daegu 41068, Korea. 5German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany. 6Department of Biomedical Science and Technology, Konkuk University, Seoul 143-701, Korea. 7Department of Anatomy, Konkuk University, Seoul 05029, Korea. 8IBST, Konkuk University, Seoul 05029, Korea. 9SNU Dementia Research Center, Seoul National University College of Medicine, Seoul, South Korea. 10Neuramedy Co. Ltd., Seoul, South Korea. 11Present address: Nutritional Sciences and Toxicology Department, University of California Berkeley, Berkeley, CA 94720, USA. 12Present address: IPS Intellectual Property Law Firm, Seoul, Korea. 13Present address: Neuramedy Co. Ltd., Seoul, South Korea. 14These authors contributed equally: Tae-Kyung Kim, Eun-Jin Bae, Byung Chul Jung. ✉email: Received: 20 October 2021 Revised: 27 September 2022 Accepted: 5 October 2022 Published online: 6 December 2022 T.-K. Kim et al. 2149 Fluorescent dye binding assay Recombinant human or mouse α-synuclein samples were mixed with either 10 μM thioflavin T (Sigma, #T3516) solution in glycine (pH 8.5), 0.001% Sybr Green commercial stock solution (#S7563, Invitrogen, Carlsbad, CA, USA), 50 μM X-34 (Sigma, #SML1954), or 50 μM curcumin (Sigma, #C1386). After incubation at room temperature, fluorescence measurements were performed on a Synergy NEO plate reader (Biotek, Winooski, VT, USA). Excitation and emission wavelengths were set at 440 nm and 490 nm, respectively, for thioflavin T, at 485 nm and 520 nm for Sybr Green, at 380 nm and 520 nm for X-34, and at 440 nm and 519 nm for curcumin. Sedimentation assay Twenty microliters of sample (200 μM in PBS) was mixed with 280 μl of DPBS (Gibco, Carlsbad, CA, USA, #A1285601) and placed on top of 30% sucrose, with the bottom of the tube containing 5% sucrose. Samples were centrifuged at 38,000 rpm in a Beckman XL-90K ultracentrifuge using an SW-41Ti rotor (Beckman). Fractions were collected and mixed with Laemmli sample buffer. Proteinase K (PK) digestion α-Synuclein samples (5 µM) were used for PK digestion. PK digestion was performed as previously described12. The remaining band intensity after PK digestion was q (...truncated)