Parkinson’s disease-related phosphorylation at Tyr39 rearranges α-synuclein amyloid fibril structure revealed by cryo-EM

Posttranslational modifications (PTMs) of α-synuclein (α-syn), e.g., phosphorylation, play an important role in modulating α-syn pathology in Parkinson’s disease (PD) and α-synucleinopathies. Accumulation of phosphorylated α-syn fibrils in Lewy bodies and Lewy neurites is the histological hallmark of these diseases. However, it is unclear how phosphorylation relates to α-syn pathology. Here, by combining chemical synthesis and bacterial expression, we obtained homogeneous α-syn fibrils with site-specific phosphorylation at Y39, which exhibits enhanced neuronal pathology in rat primary cortical neurons. We determined the cryo-electron microscopy (cryo-EM) structure of the pY39 α-syn fibril, which reveals a fold of α-syn with pY39 in the center of the fibril core forming an electrostatic interaction network with eight charged residues in the N-terminal region of α-syn. This structure composed of residues 1 to 100 represents the largest α-syn fibril core determined so far. This work provides structural understanding on the pathology of the pY39 α-syn fibril and highlights the importance of PTMs in defining the polymorphism and pathology of amyloid fibrils in neurodegenerative diseases.

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Parkinson’s disease-related phosphorylation at Tyr39 rearranges α-synuclein amyloid fibril structure revealed by cryo-EM

10.1101/2020.04.13.040261 ◽

2020 ◽

Author(s):

Kun Zhao ◽

Yeh-Jun Lim ◽

Zhenying Liu ◽

Houfang Long ◽

Yunpeng Sun ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Cortical Neurons ◽

Amyloid Fibrils ◽

Amyloid Fibril ◽

Lewy Bodies ◽

Interaction Network ◽

Lewy Neurites ◽

Post Translational Modifications ◽

Fibril Structure

AbstractPost-translational modifications (PTMs) of α-synuclein (α-syn), e.g. phosphorylation, play an important role in modulating α-syn pathology in Parkinson’s disease (PD) and α-synucleinopathies. Accumulation of phosphorylated α-syn fibrils in Lewy bodies and Lewy neurites is the histological hallmark of these diseases. However, it is unclear how phosphorylation relates to α-syn pathology. Here, by combining chemical synthesis and bacterial expression, we obtained homogeneous α-syn fibrils with site-specific phosphorylation at Y39, which exhibits enhanced neuronal pathology in rat primary cortical neurons. We determined the cryo-EM structure of pY39 α-syn fibril, which reveals a new fold of α-syn with pY39 in the center of the fibril core forming electrostatic interaction network with eight charged residues in the N-terminal region of α-syn. This structure composed of residues 1-100 represents the largest α-syn fibril core determined so far. This work provides structural understanding on the pathology of pY39 α-syn fibril, and highlights the importance of PTMs in defining the polymorphism and pathology of amyloid fibrils in neurodegenerative diseases.

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Cryo-EM structure of alpha-synuclein fibrils

eLife ◽

10.7554/elife.36402 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 188

Author(s):

Ricardo Guerrero-Ferreira ◽

Nicholas MI Taylor ◽

Daniel Mona ◽

Philippe Ringler ◽

Matthias E Lauer ◽

...

Keyword(s):

Electron Microscopy ◽

Parkinson’S Disease ◽

Parkinson's Disease ◽

Lewy Bodies ◽

Alpha Synuclein ◽

Diagnosis And Treatment ◽

Lewy Neurites ◽

Cryo Electron Microscopy ◽

High Concentrations ◽

Hydrophobic Cleft

Parkinson’s disease is a progressive neuropathological disorder that belongs to the class of synucleinopathies, in which the protein alpha-synuclein is found at abnormally high concentrations in affected neurons. Its hallmark are intracellular inclusions called Lewy bodies and Lewy neurites. We here report the structure of cytotoxic alpha-synuclein fibrils (residues 1–121), determined by cryo-electron microscopy at a resolution of 3.4 Å. Two protofilaments form a polar fibril composed of staggered β-strands. The backbone of residues 38 to 95, including the fibril core and the non-amyloid component region, are well resolved in the EM map. Residues 50–57, containing three of the mutation sites associated with familial synucleinopathies, form the interface between the two protofilaments and contribute to fibril stability. A hydrophobic cleft at one end of the fibril may have implications for fibril elongation, and invites for the design of molecules for diagnosis and treatment of synucleinopathies.

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Fibroblast Growth Factor 20 Gene Polymorphism in Parkinson’s Disease in Asian Population: A Meta-Analysis

Genes ◽

10.3390/genes12050674 ◽

2021 ◽

Vol 12 (5) ◽

pp. 674

Author(s):

Han-Lin Chiang ◽

Yih-Ru Wu ◽

Yi-Chun Chen ◽

Hon-Chung Fung ◽

Chiung-Mei Chen

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Meta Analysis ◽

Lewy Bodies ◽

Asian Population ◽

Protective Role ◽

Lewy Neurites ◽

Chinese Populations ◽

Study Results

Parkinson’s disease (PD) is a neurodegenerative disease with the pathological hallmark of Lewy bodies and Lewy neurites composed of α-synuclein. The SNP rs591323 is one of the risk loci located near the FGF20 gene that has been implicated in PD. The variation of FGF20 in the 3′ untranslated region was shown to increase α-synuclein expression. We examined the association of rs591323 with the risk of PD in a Taiwanese population and conducted a meta-analysis, including our study and two other studies from China, to further confirm the role of this SNP in Taiwanese/Chinese populations. A total of 586 patients with PD and 586 health controls (HCs) were included in our study. We found that the minor allele (A) and the AA + GA genotype under the dominant model are significantly less frequent in PD than in controls. The meta-analysis consisted of 1950 patients with PD and 2073 healthy controls from three studies. There was significant association between rs591323 and the risk of PD in the additive (Z = −3.96; p < 0.0001) and the dominant models (Z = −4.01; p < 0.0001). Our study results and the meta-analysis support the possible protective role of the rs591323 A allele in PD in Taiwanese/Chinese populations.

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Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjaa075 ◽

2021 ◽

Author(s):

Maarten C Hardenberg ◽

Tessa Sinnige ◽

Sam Casford ◽

Samuel Dada ◽

Chetan Poudel ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Self Assembly ◽

Lewy Body ◽

Amyloid Fibrils ◽

Liquid Droplet ◽

Large Body ◽

Lewy Bodies ◽

Cellular Components

Abstract Misfolded α-synuclein is a major component of Lewy bodies, which are a hallmark of Parkinson’s disease. A large body of evidence shows that α-synuclein can aggregate into amyloid fibrils, but the relationship between α-synuclein self-assembly and Lewy body formation remains unclear. Here we show, both in vitro and in a Caenorhabditis elegans model of Parkinson’s disease, that α-synuclein undergoes liquid‒liquid phase separation by forming a liquid droplet state, which converts into an amyloid-rich hydrogel with Lewy-body-like properties. This maturation process towards the amyloid state is delayed in the presence of model synaptic vesicles in vitro. Taken together, these results suggest that the formation of Lewy bodies may be linked to the arrested maturation of α-synuclein condensates in the presence of lipids and other cellular components.

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Parkinson’s disease is a type of amyloidosis featuring accumulation of amyloid fibrils of α-synuclein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906124116 ◽

2019 ◽

Vol 116 (36) ◽

pp. 17963-17969 ◽

Cited By ~ 24

Author(s):

Katsuya Araki ◽

Naoto Yagi ◽

Koki Aoyama ◽

Chi-Jing Choong ◽

Hideki Hayakawa ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Amyloid Fibrils ◽

Lewy Bodies ◽

X Ray Diffraction ◽

Thin Sections ◽

X Ray ◽

Β Sheet ◽

The Brain

Many neurodegenerative diseases are characterized by the accumulation of abnormal protein aggregates in the brain. In Parkinson’s disease (PD), α-synuclein (α-syn) forms such aggregates called Lewy bodies (LBs). Recently, it has been reported that aggregates of α-syn with a cross-β structure are capable of propagating within the brain in a prionlike manner. However, the presence of cross-β sheet-rich aggregates in LBs has not been experimentally demonstrated so far. Here, we examined LBs in thin sections of autopsy brains of patients with PD using microbeam X-ray diffraction (XRD) and found that some of them gave a diffraction pattern typical of a cross-β structure. This result confirms that LBs in the brain of PD patients contain amyloid fibrils with a cross-β structure and supports the validity of in vitro propagation experiments using artificially formed amyloid fibrils of α-syn. Notably, our finding supports the concept that PD is a type of amyloidosis, a disease featuring the accumulation of amyloid fibrils of α-syn.

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C-terminal α-synuclein truncations are linked to cysteine cathepsin activity in Parkinson's disease

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.008930 ◽

2019 ◽

Vol 294 (25) ◽

pp. 9973-9984 ◽

Cited By ~ 11

Author(s):

Ryan P. McGlinchey ◽

Shannon M. Lacy ◽

Katherine E. Huffer ◽

Nahid Tayebi ◽

Ellen Sidransky ◽

...

Keyword(s):

Parkinson’S Disease ◽

Amino Acids ◽

Parkinson's Disease ◽

Amyloid Fibrils ◽

Lewy Bodies ◽

Amyloid Formation ◽

Pathological Feature ◽

Cysteine Cathepsins ◽

Cathepsin Activity ◽

Cysteine Cathepsin

A pathological feature of Parkinson's disease (PD) is Lewy bodies (LBs) composed of α-synuclein (α-syn) amyloid fibrils. α-Syn is a 140 amino acids–long protein, but truncated α-syn is enriched in LBs. The proteolytic processes that generate these truncations are not well-understood. On the basis of our previous work, we propose that these truncations could originate from lysosomal activity attributable to cysteine cathepsins (Cts). Here, using a transgenic SNCAA53T mouse model, overexpressing the PD-associated α-syn variant A53T, we compared levels of α-syn species in purified brain lysosomes from nonsymptomatic mice with those in age-matched symptomatic mice. In the symptomatic mice, antibody epitope mapping revealed enrichment of C-terminal truncations, resulting from CtsB, CtsL, and asparagine endopeptidase. We did not observe changes in individual cathepsin activities, suggesting that the increased levels of C-terminal α-syn truncations are because of the burden of aggregated α-syn. Using LC-MS and purified α-syn, we identified C-terminal truncations corresponding to amino acids 1–122 and 1–90 from the SNCAA53T lysosomes. Feeding rat dopaminergic N27 cells with exogenous α-syn fibrils confirmed that these fragments originate from incomplete fibril degradation in lysosomes. We mimicked these events in situ by asparagine endopeptidase degradation of α-syn fibrils. Importantly, the resulting C-terminally truncated fibrils acted as superior seeds in stimulating α-syn aggregation compared with that of the full-length fibrils. These results unequivocally show that C-terminal α-syn truncations in LBs are linked to Cts activities, promote amyloid formation, and contribute to PD pathogenesis.

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LRRK2 at the Crossroad of Aging and Parkinson’s Disease

Genes ◽

10.3390/genes12040505 ◽

2021 ◽

Vol 12 (4) ◽

pp. 505

Author(s):

Eun-Mi Hur ◽

Byoung Dae Lee

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Late Onset ◽

Lewy Bodies ◽

Substantia Nigra Pars Compacta ◽

Cellular Functions ◽

Lewy Neurites ◽

Clinical Presentations ◽

Lrrk2 Gene ◽

Cellular Dysfunction

Parkinson’s disease (PD) is a heterogeneous neurodegenerative disease characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the widespread occurrence of proteinaceous inclusions known as Lewy bodies and Lewy neurites. The etiology of PD is still far from clear, but aging has been considered as the highest risk factor influencing the clinical presentations and the progression of PD. Accumulating evidence suggests that aging and PD induce common changes in multiple cellular functions, including redox imbalance, mitochondria dysfunction, and impaired proteostasis. Age-dependent deteriorations in cellular dysfunction may predispose individuals to PD, and cellular damages caused by genetic and/or environmental risk factors of PD may be exaggerated by aging. Mutations in the LRRK2 gene cause late-onset, autosomal dominant PD and comprise the most common genetic causes of both familial and sporadic PD. LRRK2-linked PD patients show clinical and pathological features indistinguishable from idiopathic PD patients. Here, we review cellular dysfunctions shared by aging and PD-associated LRRK2 mutations and discuss how the interplay between the two might play a role in PD pathologies.

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Mitochondrial Dysfunction, Protein Misfolding and Neuroinflammation in Parkinson’s Disease: Roads to Biomarker Discovery

Biomolecules ◽

10.3390/biom11101508 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1508

Author(s):

Anna Picca ◽

Flora Guerra ◽

Riccardo Calvani ◽

Roberta Romano ◽

Hélio José Coelho-Júnior ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Mitochondrial Dysfunction ◽

Protein Misfolding ◽

Biomarker Discovery ◽

Lewy Bodies ◽

Substantia Nigra Pars Compacta ◽

Lewy Neurites ◽

Post Translational Modifications ◽

Ubiquitin Proteasome

Parkinson’s Disease (PD) is a highly prevalent neurodegenerative disease among older adults. PD neuropathology is marked by the progressive loss of the dopaminergic neurons of the substantia nigra pars compacta and the widespread accumulation of misfolded intracellular α-synuclein (α-syn). Genetic mutations and post-translational modifications, such as α-syn phosphorylation, have been identified among the multiple factors supporting α-syn accrual during PD. A decline in the clearance capacity of the ubiquitin-proteasome and the autophagy-lysosomal systems, together with mitochondrial dysfunction, have been indicated as major pathophysiological mechanisms of PD neurodegeneration. The accrual of misfolded α-syn aggregates into soluble oligomers, and the generation of insoluble fibrils composing the core of intraneuronal Lewy bodies and Lewy neurites observed during PD neurodegeneration, are ignited by the overproduction of reactive oxygen species (ROS). The ROS activate the α-syn aggregation cascade and, together with the Lewy bodies, promote neurodegeneration. However, the molecular pathways underlying the dynamic evolution of PD remain undeciphered. These gaps in knowledge, together with the clinical heterogeneity of PD, have hampered the identification of the biomarkers that may be used to assist in diagnosis, treatment monitoring, and prognostication. Herein, we illustrate the main pathways involved in PD pathogenesis and discuss their possible exploitation for biomarker discovery.

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Srpk3 Decrease Associated with Alpha-Synuclein Increase in Muscles of MPTP-Induced Parkinson’s Disease Mice

International Journal of Molecular Sciences ◽

10.3390/ijms22179375 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9375

Author(s):

Min Hyung Seo ◽

Sujung Yeo

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

C2c12 Cell ◽

Lewy Bodies ◽

Alpha Synuclein ◽

C2c12 Cells ◽

Control Group ◽

Lewy Neurites ◽

Lewy Pathology ◽

Interfering Rna

Parkinson’s disease (PD) is characterized by a loss of dopaminergic cells in the substantia nigra, and its histopathological features include the presence of fibrillar aggregates of α-synuclein (α-syn), which are called Lewy bodies and Lewy neurites. Lewy pathology has been identified not only in the brain but also in various tissues, including muscles. This study aimed to investigate the link between serine/arginine-rich protein specific kinase 3 (srpk3) and α-syn in muscles in PD. We conducted experiments on the quadriceps femoris of a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and the C2C12 cell line after treatment with 1-methyl-4-phenylpyridinium (MPP+) and srpk3 short interfering RNA (siRNA). Compared to the control group, the MPTP group showed significantly reduced expression of srpk3, but increased expression of α-syn. In MPP+-treated C2C12 cells, srpk3 expression gradually decreased and α-syn expression increased with the increasing MPP+ concentration. Moreover, experiments in C2C12 cells using srpk3 siRNA showed increased expressions of α-syn and phosphorylated α-syn. Our results showed that srpk3 expression could be altered by MPTP intoxication in muscles, and this change may be related to changes in α-syn expression. Furthermore, this study could contribute to advancement of research on the mechanism by which srpk3 plays a role in PD.

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Identification of a highly neurotoxic α-synuclein species inducing mitochondrial damage and mitophagy in Parkinson’s disease

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1713849115 ◽

2018 ◽

Vol 115 (11) ◽

pp. E2634-E2643 ◽

Cited By ~ 87

Author(s):

Diego Grassi ◽

Shannon Howard ◽

Minghai Zhou ◽

Natalia Diaz-Perez ◽

Nicolai T. Urban ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Lewy Bodies ◽

Stimulated Emission ◽

Primary Neurons ◽

Mitochondrial Fragmentation ◽

Cytochrome C Release ◽

Lewy Neurites ◽

Western Blots ◽

Mitochondrial Membrane Depolarization

Exposure of cultured primary neurons to preformed α-synuclein fibrils (PFFs) leads to the recruitment of endogenous α-synuclein and its templated conversion into fibrillar phosphorylated α-synuclein (pα-synF) aggregates resembling those involved in Parkinson’s disease (PD) pathogenesis. Pα-synF was described previously as inclusions morphologically similar to Lewy bodies and Lewy neurites in PD patients. We discovered the existence of a conformationally distinct, nonfibrillar, phosphorylated α-syn species that we named “pα-syn*.” We uniquely describe the existence of pα-syn* in PFF-seeded primary neurons, mice brains, and PD patients’ brains. Through immunofluorescence and pharmacological manipulation we showed that pα-syn* results from incomplete autophagic degradation of pα-synF. Pα-synF was decorated with autophagic markers, but pα-syn* was not. Western blots revealed that pα-syn* was N- and C-terminally trimmed, resulting in a 12.5-kDa fragment and a SDS-resistant dimer. After lysosomal release, pα-syn* aggregates associated with mitochondria, inducing mitochondrial membrane depolarization, cytochrome C release, and mitochondrial fragmentation visualized by confocal and stimulated emission depletion nanoscopy. Pα-syn* recruited phosphorylated acetyl-CoA carboxylase 1 (ACC1) with which it remarkably colocalized. ACC1 phosphorylation indicates low ATP levels, AMPK activation, and oxidative stress and induces mitochondrial fragmentation via reduced lipoylation. Pα-syn* also colocalized with BiP, a master regulator of the unfolded protein response and a resident protein of mitochondria-associated endoplasmic reticulum membranes that are sites of mitochondrial fission and mitophagy. Pα-syn* aggregates were found in Parkin-positive mitophagic vacuoles and imaged by electron microscopy. Collectively, we showed that pα-syn* induces mitochondrial toxicity and fission, energetic stress, and mitophagy, implicating pα-syn* as a key neurotoxic α-syn species and a therapeutic target.

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