Cryo-EM structure of an amyloid fibril formed by full-length human SOD1 reveals its pathological conformational conversion

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by the selective death of motor neurons. Misfolded Cu, Zn-superoxide dismutase (SOD1) has been linked to both familial ALS and sporadic ALS. SOD1 fibrils formed in vitro are able to incorporate into cells, transmit intercellularly, and share toxic properties with ALS inclusions. Here we produced amyloid fibrils in vitro from recombinant, full-length apo human SOD1 under semi-reducing conditions and determined the atomic structure using cryo-EM. The SOD1 fibril consists of a single protofibril with a left-handed helix. The fibril core exhibits a serpentine fold comprising N-terminal segment (residues 3 to 55) and C-terminal segment (residues 86 to 153) with a structural break. The two segments are zipped up by three salt bridge pairs. By comparison with the structure of apo SOD1 dimer, we propose that eight β-strands (to form a β-barrel) and one α-helix in the subunit of apo SOD1 convert into thirteen β-strands stabilized by five hydrophobic cavities in the SOD1 fibril. Our data provide insights into how SOD1 converts between structurally and functionally distinct states.

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The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis

Science Translational Medicine ◽

10.1126/scitranslmed.aaf3962 ◽

2017 ◽

Vol 9 (391) ◽

pp. eaaf3962 ◽

Cited By ~ 90

Author(s):

Keiko Imamura ◽

Yuishin Izumi ◽

Akira Watanabe ◽

Kayoko Tsukita ◽

Knut Woltjen ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Motor Neuron ◽

Motor Neurons ◽

New Drugs ◽

Small Interfering Rnas ◽

Altered Expression ◽

Sporadic Als ◽

Induced Pluripotent ◽

Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS), a fatal disease causing progressive loss of motor neurons, still has no effective treatment. We developed a phenotypic screen to repurpose existing drugs using ALS motor neuron survival as readout. Motor neurons were generated from induced pluripotent stem cells (iPSCs) derived from an ALS patient with a mutation in superoxide dismutase 1 (SOD1). Results of the screen showed that more than half of the hits targeted the Src/c-Abl signaling pathway. Src/c-Abl inhibitors increased survival of ALS iPSC-derived motor neurons in vitro. Knockdown of Src or c-Abl with small interfering RNAs (siRNAs) also rescued ALS motor neuron degeneration. One of the hits, bosutinib, boosted autophagy, reduced the amount of misfolded mutant SOD1 protein, and attenuated altered expression of mitochondrial genes. Bosutinib also increased survival in vitro of ALS iPSC-derived motor neurons from patients with sporadic ALS or other forms of familial ALS caused by mutations in TAR DNA binding protein (TDP-43) or repeat expansions in C9orf72. Furthermore, bosutinib treatment modestly extended survival of a mouse model of ALS with an SOD1 mutation, suggesting that Src/c-Abl may be a potentially useful target for developing new drugs to treat ALS.

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Pathological Roles of Wild-Type Cu, Zn-Superoxide Dismutase in Amyotrophic Lateral Sclerosis

Neurology Research International ◽

10.1155/2012/323261 ◽

2012 ◽

Vol 2012 ◽

pp. 1-6 ◽

Cited By ~ 11

Author(s):

Yoshiaki Furukawa

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Superoxide Dismutase ◽

Motor Neurons ◽

Wild Type ◽

Sporadic Als ◽

Familial Form ◽

Recent Developments ◽

Lateral Sclerosis

Dominant mutations in a Cu, Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (ALS). While it remains controversial how SOD1 mutations lead to onset and progression of the disease, manyin vitroandin vivostudies have supported a gain-of-toxicity mechanism where pathogenic mutations contribute to destabilizing a native structure of SOD1 and thus facilitate misfolding and aggregation. Indeed, abnormal accumulation of SOD1-positive inclusions in spinal motor neurons is a pathological hallmark in SOD1-related familial ALS. Furthermore, similarities in clinical phenotypes and neuropathology of ALS cases with and without mutations insod1gene have implied a disease mechanism involving SOD1 common to all ALS cases. Although pathogenic roles of wild-type SOD1 in sporadic ALS remain controversial, recent developments of novel SOD1 antibodies have made it possible to characterize wild-type SOD1 under pathological conditions of ALS. Here, I have briefly reviewed recent progress on biochemical and immunohistochemical characterization of wild-type SOD1 in sporadic ALS cases and discussed possible involvement of wild-type SOD1 in a pathomechanism of ALS.

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Coactivation of GSK3β and IGF-1 Attenuates Amyotrophic Lateral Sclerosis Nerve Fiber Cytopathies in SOD1 Mutant Patient-Derived Motor Neurons

Cells ◽

10.3390/cells10102773 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2773

Author(s):

Hsiao-Chien Ting ◽

Hui-I Yang ◽

Horng-Jyh Harn ◽

Ing-Ming Chiu ◽

Hong-Lin Su ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Nerve Fiber ◽

Motor Neurons ◽

Induced Pluripotent Stem Cell ◽

Therapeutic Effects ◽

Patient Death ◽

Sporadic Als ◽

Sod1 Mutant ◽

Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive nervous system disease that causes motor neuron (MN) degeneration and results in patient death within a few years. To recapitulate the cytopathies of ALS patients’ MNs, SOD1G85R mutant and corrected SOD1G85G isogenic-induced pluripotent stem cell (iPSC) lines were established. Two SOD1 mutant ALS (SOD1G85R and SOD1D90A), two SOD1 mutant corrected (SOD1G85G and SOD1D90D), and one sporadic ALS iPSC lines were directed toward MNs. After receiving ~90% purity for MNs, we first demonstrated that SOD1G85R mutant ALS MNs recapitulated ALS-specific nerve fiber aggregates, similar to SOD1D90A ALS MNs in a previous study. Moreover, we found that both SOD1 mutant MNs showed ALS-specific neurite degenerations and neurotransmitter-induced calcium hyperresponsiveness. In a small compound test using these MNs, we demonstrated that gastrodin, a major ingredient of Gastrodia elata, showed therapeutic effects that decreased nerve fiber cytopathies and reverse neurotransmitter-induced hyperresponsiveness. The therapeutic effects of gastrodin applied not only to SOD1 ALS MNs but also to sporadic ALS MNs and SOD1G93A ALS mice. Moreover, we found that coactivation of the GSK3β and IGF-1 pathways was a mechanism involved in the therapeutic effects of gastrodin. Thus, the coordination of compounds that activate these two mechanisms could reduce nerve fiber cytopathies in SOD1 ALS MNs. Interestingly, the therapeutic role of GSK3β activation on SOD1 ALS MNs in the present study was in contrast to the role previously reported in research using cell line- or transgenic animal-based models. In conclusion, we identified in vitro ALS-specific nerve fiber and neurofunctional markers in MNs, which will be useful for drug screening, and we used an iPSC-based model to reveal novel therapeutic mechanisms (including GSK3β and IGF-1 activation) that may serve as potential targets for ALS therapy.

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Activation of brain calcineurin (Cn) by Cu-Zn superoxide dismutase (SOD1) depends on direct SOD1–Cn protein interactions occurring in vitro and in vivo

Biochemical Journal ◽

10.1042/bj20061202 ◽

2007 ◽

Vol 405 (1) ◽

pp. 51-59 ◽

Cited By ~ 15

Author(s):

Abdulbaki Agbas ◽

Dongwei Hui ◽

Xinsheng Wang ◽

Vekalet Tek ◽

Asma Zaidi ◽

...

Keyword(s):

Superoxide Dismutase ◽

Protein Interactions ◽

Active Site ◽

Conformational State ◽

Full Length ◽

Poor Correlation ◽

Calcineurin Activity ◽

Human Sod1

Cn (calcineurin) activity is stabilized by SOD1 (Cu-Zn superoxide dismutase), a phenomenon attributed to protection from superoxide (O2•−). The effects of O2•− on Cn are still controversial. We found that O2•−, generated either in vitro or in vivo did not affect Cn activity. Yet native bovine, recombinant human or rat, and two chimaeras of human SOD1–rat SOD1, all activated Cn, but SOD2 (Mn-superoxide dismutase) did not affect Cn activity. There was also a poor correlation between SOD1 dismutase activity and Cn activation. A chimaera of human N-terminal SOD1 and rat C-terminal SOD1 had little detectable dismutase activity, yet stimulated Cn activity the same as full-length human or rat SOD1. Nevertheless, there was evidence that the active site of SOD1 was involved in Cn activation based on the loss of activation following chelation of Cu from the active site of SOD1. Also, SOD1 engaged in the catalysis of O2•− dismutation was ineffective in activating Cn. SOD1 activation of Cn resulted from a 90-fold decrease in phosphatase Km without a change in Vmax. A possible mechanism for the activation of Cn was identified in our studies as the prevention of Fe and Zn losses from the active site of Cn, suggesting a conformation-dependent SOD1–Cn interaction. In neurons, SOD1 and Cn were co-localized in cytoplasm and membranes, and SOD1 co-immunoprecipitated with Cn from homogenates of brain hippocampus and was present in immunoprecipitates as large multimers. Pre-incubation of pure SOD1 with Cn caused SOD1 multimer formation, an indication of an altered conformational state in SOD1 upon interaction with Cn.

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Overexpression of ferroptosis defense enzyme Gpx4 retards motor neuron disease of SOD1G93A mice

Scientific Reports ◽

10.1038/s41598-021-92369-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Liuji Chen ◽

Ren Na ◽

Kirsten Danae McLane ◽

Cody Sylvester Thompson ◽

Ju Gao ◽

...

Keyword(s):

Lipid Peroxidation ◽

Transgenic Mice ◽

Motor Neuron ◽

Motor Neuron Disease ◽

Motor Neurons ◽

Disease Onset ◽

Motor Neuron Degeneration ◽

Neuron Degeneration ◽

Sporadic Als

AbstractDegeneration and death of motor neurons in Amyotrophic Lateral Sclerosis (ALS) are associated with increased lipid peroxidation. Lipid peroxidation is the driver of ferroptosis, an iron-dependent oxidative mode of cell death. However, the importance of ferroptosis in motor neuron degeneration of ALS remains unclear. Glutathione peroxidase 4 (Gpx4) is a key enzyme in suppressing ferroptosis by reducing phospholipid hydroperoxides in membranes. To assess the effect of increased protection against ferroptosis on motor neuron disease, we generated SOD1G93AGPX4 double transgenic mice by cross-breeding GPX4 transgenic mice with SOD1G93A mice, a widely used ALS mouse model. Compared with control SOD1G93A mice, both male and female SOD1G93AGPX4 mice had extended lifespans. SOD1G93AGPX4 mice also showed delayed disease onset and increased motor function, which were correlated with ameliorated spinal motor neuron degeneration and reduced lipid peroxidation. Moreover, cell toxicity induced by SOD1G93A was ameliorated by Gpx4 overexpression and by chemical inhibitors of ferroptosis in vitro. We further found that the anti-ferroptosis defense system in spinal cord tissues of symptomatic SOD1G93A mice and sporadic ALS patients might be compromised due to deficiency of Gpx4. Thus, our results suggest that ferroptosis plays a key role in motor neuron degeneration of ALS.

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Nanostructure and stability of calcitonin amyloids

Journal of Biological Chemistry ◽

10.1074/jbc.m116.770271 ◽

2017 ◽

Vol 292 (18) ◽

pp. 7348-7357 ◽

Cited By ~ 8

Author(s):

Federica Rigoldi ◽

Pierangelo Metrangolo ◽

Alberto Redaelli ◽

Alfonso Gautieri

Keyword(s):

Amyloid Fibrils ◽

Md Simulations ◽

Full Length ◽

Structural Basis ◽

Human Calcitonin ◽

Nmr Data ◽

Parallel Arrangement ◽

Β Sheets ◽

Α Helix ◽

Β Sheet

Calcitonin is a 32-amino acid thyroid hormone that can form amyloid fibrils. The structural basis of the fibril formation and stabilization is still debated and poorly understood. The reason is that NMR data strongly suggest antiparallel β-sheet calcitonin assembly, whereas modeling studies on the short DFNKF peptide (corresponding to the sequence from Asp15 to Phe19 of human calcitonin and reported as the minimal amyloidogenic module) show that it assembles with parallel β-sheets. In this work, we first predict the structure of human calcitonin through two complementary molecular dynamics (MD) methods, finding that human calcitonin forms an α-helix. We use extensive MD simulations to compare previously proposed calcitonin fibril structures. We find that two conformations, the parallel arrangement and one of the possible antiparallel structures (with Asp15 and Phe19 aligned), are highly stable and ordered. Nonetheless, fibrils with parallel molecules show bulky loops formed by residues 1 to 7 located on the same side, which could limit or prevent the formation of larger amyloids. We investigate fibrils formed by the DFNKF peptide by simulating different arrangements of this amyloidogenic core sequence. We show that DFNKF fibrils are highly stable when assembled in parallel β-sheets, whereas they quickly unfold in antiparallel conformation. Our results indicate that the DFNKF peptide represents only partially the full-length calcitonin behavior. Contrary to the full-length polypeptide, in fact, the DFNKF sequence is not stable in antiparallel conformation, suggesting that the residue flanking the amyloidogenic peptide contributes to the stabilization of the experimentally observed antiparallel β-sheet packing.

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Cryo-EM structure and inhibitor design of human IAPP (amylin) fibrils

10.1101/2020.05.25.114967 ◽

2020 ◽

Author(s):

Qin Cao ◽

David R. Boyer ◽

Michael R. Sawaya ◽

Peng Ge ◽

David S. Eisenberg

Keyword(s):

Amyloid Fibrils ◽

Full Length ◽

Hydrophobic Core ◽

Inhibitor Design ◽

Islet Amyloid ◽

Fibril Structure ◽

Starting Point ◽

High Resolution Structure ◽

Aggregation Inhibitors

AbstractHuman islet amyloid polypeptide (hIAPP, or amylin) is a 37 amino acid hormone secreted by pancreatic islet β-cells. Aggregation of hIAPP into amyloid fibrils is found in more than 90% of Type-II Diabetes (T2D) patients and is considered to be associated with T2D pathology. Although different models have been proposed, the high resolution structure of hIAPP fibrils is unknown. Here we report the cryo-EM structure of recombinant full-length hIAPP fibrils. The fibril is composed of two symmetrically-related protofilaments with ordered residues 14-37 that meet at a 14-residue central hydrophobic core. Our hIAPP fibril structure (i) supports the previous hypothesis that residues 20-29, especially 23-29 are the primary amyloid core of hIAPP, (ii) suggests a molecular mechanism for the action of the hIAPP hereditary mutation S20G, (iii) explains why the 6 residue substitutions in rodent IAPP prevent aggregation, and (iv) suggests possible regions responsible for the observed hIAPP cross-seeding with β-amyloid. Furthermore, we performed structure-based inhibitor design to generate potential hIAPP aggregation inhibitors via a capping strategy. Four of the designed candidates delay hIAPP aggregation in vitro, providing a starting point for the development of T2D therapeutics and proof-of-concept that the capping strategy can be used on full-length cryo-EM fibril structures.

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In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162624 ◽

1996 ◽

Vol 54 ◽

pp. 32-33

Author(s):

C. Jennermann ◽

S. A. Kliewer ◽

D. C. Morris

Keyword(s):

Alkaline Phosphatase ◽

Room Temperature ◽

Uncoupling Protein ◽

Ppar Gamma ◽

Nuclear Hormone Receptor ◽

Full Length ◽

Northern Analysis ◽

Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

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Global transcriptome profile of the developmental principles of in vitro iPSC-to-motor neuron differentiation

BMC Molecular and Cell Biology ◽

10.1186/s12860-021-00343-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Emilia Solomon ◽

Katie Davis-Anderson ◽

Blake Hovde ◽

Sofiya Micheva-Viteva ◽

Jennifer Foster Harris ◽

...

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Motor Neuron ◽

Motor Neurons ◽

Acetylcholine Receptors ◽

Gene Networks ◽

Spinal Cord Injuries ◽

Regulatory Gene ◽

Transcriptome Profile

Abstract Background Human induced pluripotent stem cells (iPSC) have opened new avenues for regenerative medicine. Consequently, iPSC-derived motor neurons have emerged as potentially viable therapies for spinal cord injuries and neurodegenerative disorders including Amyotrophic Lateral Sclerosis. However, direct clinical application of iPSC bears in itself the risk of tumorigenesis and other unforeseeable genetic or epigenetic abnormalities. Results Employing RNA-seq technology, we identified and characterized gene regulatory networks triggered by in vitro chemical reprogramming of iPSC into cells with the molecular features of motor neurons (MNs) whose function in vivo is to innervate effector organs. We present meta-transcriptome signatures of 5 cell types: iPSCs, neural stem cells, motor neuron progenitors, early motor neurons, and mature motor neurons. In strict response to the chemical stimuli, along the MN differentiation axis we observed temporal downregulation of tumor growth factor-β signaling pathway and consistent activation of sonic hedgehog, Wnt/β-catenin, and Notch signaling. Together with gene networks defining neuronal differentiation (neurogenin 2, microtubule-associated protein 2, Pax6, and neuropilin-1), we observed steady accumulation of motor neuron-specific regulatory genes, including Islet-1 and homeobox protein HB9. Interestingly, transcriptome profiling of the differentiation process showed that Ca2+ signaling through cAMP and LPC was downregulated during the conversion of the iPSC to neural stem cells and key regulatory gene activity of the pathway remained inhibited until later stages of motor neuron formation. Pathways shaping the neuronal development and function were well-represented in the early motor neuron cells including, neuroactive ligand-receptor interactions, axon guidance, and the cholinergic synapse formation. A notable hallmark of our in vitro motor neuron maturation in monoculture was the activation of genes encoding G-coupled muscarinic acetylcholine receptors and downregulation of the ionotropic nicotinic acetylcholine receptors expression. We observed the formation of functional neuronal networks as spontaneous oscillations in the extracellular action potentials recorded on multi-electrode array chip after 20 days of differentiation. Conclusions Detailed transcriptome profile of each developmental step from iPSC to motor neuron driven by chemical induction provides the guidelines to novel therapeutic approaches in the re-construction efforts of muscle innervation.

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