bims-proned 2024-10-06 papers

bims-proned

Biomed News

on Proteostasis in neurodegeneration

Issue of 2024‒10‒06
twelve papers selected by
Verena Kohler, Umeå University

Pathological Involvement of Protein Phase Separation and Aggregation in Neurodegenerative Diseases.
Biophysical Studies of Amyloid-Binding Fluorophores to Tau AD Core Fibrils Formed without Cofactors.
Aggregation of the amyloid-β peptide (Aβ40) within condensates generated through liquid-liquid phase separation.
Vanillin Mitigates the MPTP-Induced α-Synucleinopathy in a Mouse Model of Parkinson's Disease: Insights into the Involvement of Wnt/β-Catenin Signaling.
High-Resolution Cryo-EM Structure Determination of α-Synuclein - A Prototypical Amyloid Fibril.
From Brain to Muscle: The Role of Muscle Tissue in Neurodegenerative Disorders.
Alpha-synuclein preformed fibril-induced aggregation and dopaminergic cell death in cathepsin D overexpression and ZKSCAN3 knockout mice.
A novel peptide-based tau aggregation inhibitor as a potential therapeutic for Alzheimer's disease and other tauopathies.
Targeting the TDP-43 low complexity domain blocks spreading of pathology in a mouse model of ALS/FTD.
Exploring the Role of Reactive Oxygen Species in the Pathogenesis and Pathophysiology of Alzheimer's and Parkinson's Disease and the Efficacy of Antioxidant Treatment.
Capturing the illusive ring-shaped intermediates in Aβ42 amyloid formation.
TDP-43 Amyloid Fibril Formation via Phase Separation-Related and -Unrelated Pathways.

Int J Mol Sci. 2024 Sep 23. pii: 10187. [Epub ahead of print]25(18):

Pathological Involvement of Protein Phase Separation and Aggregation in Neurodegenerative Diseases.

Yinuo Wu, Biao Ma, Chang Liu, Dangdang Li, Guangchao Sui.

  Neurodegenerative diseases are the leading cause of human disability and immensely reduce patients' life span and quality. The diseases are characterized by the functional loss of neuronal cells and share several common pathogenic mechanisms involving the malfunction, structural distortion, or aggregation of multiple key regulatory proteins. Cellular phase separation is the formation of biomolecular condensates that regulate numerous biological processes, including neuronal development and synaptic signaling transduction. Aberrant phase separation may cause protein aggregation that is a general phenomenon in the neuronal cells of patients suffering neurodegenerative diseases. In this review, we summarize the pathological causes of common neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, among others. We discuss the regulation of key amyloidogenic proteins with an emphasis of their aberrant phase separation and aggregation. We also introduce the approaches as potential therapeutic strategies to ameliorate neurodegenerative diseases through intervening protein aggregation. Overall, this review consolidates the research findings of phase separation and aggregation caused by misfolded proteins in a context of neurodegenerative diseases.

Keywords:  neurodegenerative diseases; pathology; phase separation and aggregation; treatment

DOI:  https://doi.org/10.3390/ijms251810187
Int J Mol Sci. 2024 Sep 15. pii: 9946. [Epub ahead of print]25(18):

Biophysical Studies of Amyloid-Binding Fluorophores to Tau AD Core Fibrils Formed without Cofactors.

Daniela P Freitas, Joana Saavedra, Isabel Cardoso, Cláudio M Gomes.

  Tau is an intrinsically disordered protein involved in several neurodegenerative diseases where a common hallmark is the appearance of tau aggregates in the brain. One common approach to elucidate the mechanisms behind the aggregation of tau has been to recapitulate in vitro the self-assembly process in a fast and reproducible manner. While the seeding of tau aggregation is prompted by negatively charged cofactors, the obtained fibrils are morphologically distinct from those found in vivo. The Tau AD core fragment (TADC, tau 306-378) has emerged as a new model and potential solution for the cofactor-free in vitro aggregation of tau. Here, we use TADC to further study this process combining multiple amyloid-detecting fluorophores and fibril bioimaging. We confirmed by transmission electron microscopy that this fragment forms fibrils after quiescent incubation at 37 °C. We then employed a panel of eight amyloid-binding fluorophores to query the formed species by acquiring their emission spectra. The results obtained showed that nearly all dyes detect TADC self-assembled species. However, the successful monitoring of TADC aggregation kinetics was limited to three fluorophores (X-34, Bis-ANS, and pFTAA) which yielded sigmoidal curves but different aggregation half-times, hinting to different species being detected. Altogether, this study highlights the potential of using multiple extrinsic fluorescent probes, alone or in combination, as tools to further clarify mechanisms behind the aggregation of amyloidogenic proteins.

Keywords:  amyloid-binding fluorophores; fibril aggregation kinetics; microtubule-binding repeats; tau aggregation

DOI:  https://doi.org/10.3390/ijms25189946
Sci Rep. 2024 09 30. 14(1): 22633

Aggregation of the amyloid-β peptide (Aβ40) within condensates generated through liquid-liquid phase separation.

Owen M Morris, Zenon Toprakcioglu, Alexander Röntgen, Mariana Cali, Tuomas P J Knowles, Michele Vendruscolo.

The deposition of the amyloid-β (Aβ) peptide into amyloid fibrils is a hallmark of Alzheimer's disease. Recently, it has been reported that some proteins can aggregate and form amyloids through an intermediate pathway involving a liquid-like condensed phase. These observations prompted us to investigate the phase space of Aβ. We thus explored the ability of Aβ to undergo liquid-liquid phase separation, and the subsequent liquid-to-solid transition that takes place within the resulting condensates. Through the use of microfluidic approaches, we observed that the 40-residue form of Αβ (Αβ40) can undergo liquid-liquid phase separation, and that accessing a liquid-like intermediate state enables Αβ40 to self-assemble and aggregate into amyloid fibrils through this pathway. These results prompt further studies to investigate the possible role of Αβ liquid-liquid phase separation and its subsequent aggregation in the context of Alzheimer's disease and more generally on neurodegenerative processes.

DOI: https://doi.org/10.1038/s41598-024-72265-7
J Integr Neurosci. 2024 Sep 23. 23(9): 175

Vanillin Mitigates the MPTP-Induced α-Synucleinopathy in a Mouse Model of Parkinson's Disease: Insights into the Involvement of Wnt/β-Catenin Signaling.

Linchi Rani, Amal Chandra Mondal.

  BACKGROUND: The abnormal aggregation of α-synuclein (α-syn) in the substantia nigra pars compacta (SNpc) region of the brain is characteristic of Parkinson's disease (PD), leading to the selective demise of neurons. Modifications in the post-translational processing of α-syn, phosphorylation at Ser129 in particular, are implicated in α-syn aggregation and are considered key hallmarks of PD. Furthermore, dysregulated Wnt/β-catenin signaling, influenced by glycogen synthase kinase-3 beta (GSK-3β), is implicated in PD pathogenesis. Inhibition of GSK-3β holds promise in promoting neuroprotection by enhancing the Wnt/β-catenin pathway.METHODS: In our previous study utilizing 1-methyl-4-phenylpyridinium (MPP+)-administered differentiated SH-SY5Y cells and a PD mouse model, we explored Vanillin's neuroprotective properties and related mechanisms against neuronal loss induced by MPP+/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration. In the current study, we elucidated the mitigating effects of Vanillin on motor impairments, P-Ser129-α-syn expression, Wnt/β-catenin signaling, and autophagic neuron death induced by MPTP in a mouse model of PD by performing motor function tests, western blot analysis and immunostaining.
RESULTS: Our results show that Vanillin effectively modulated the motor dysfunctions, GSK-3β expression, and activity, activated the Wnt/β-catenin signaling, and reduced autophagic neuronal demise in the MPTP-lesioned mice, highlighting its neuroprotective effects.
CONCLUSIONS: These findings underscore the complex interplay between α-syn pathology, GSK-3β, Wnt/β-catenin signaling, and autophagic-cell death in PD pathogenesis. Targeting these pathways, particularly with Vanillin, can be a promising therapeutic strategy for restoring dopaminergic (DA-ergic) neuronal homeostasis and slowing the progression of PD. Further research is crucial to resolving existing disputes and translating these discoveries into effective therapeutic interventions for PD patients.

Keywords:  Parkinson's disease; Wnt/β-catenin signaling; autophagy; neuroprotection; α-syn expression

DOI:  https://doi.org/10.31083/j.jin2309175
bioRxiv. 2024 Sep 19. pii: 2024.09.18.613698. [Epub ahead of print]

High-Resolution Cryo-EM Structure Determination of α-Synuclein - A Prototypical Amyloid Fibril.

Juan C Sanchez, Josh Pierson, Collin G Borcik, Chad M Rienstra, Elizabeth R Wright.

The physiological role of α-synuclein (α-syn), an intrinsically disordered presynaptic neuronal protein, is believed to impact the release of neurotransmitters through interactions with the SNARE complex. However, under certain cellular conditions that are not well understood, α-syn will self-assemble into β-sheet rich fibrils that accumulate and form insoluble neuronal inclusions. Studies of patient derived brain tissues have concluded that these inclusions are associated with Parkinson's disease, the second most common neurodegenerative disorder, and other synuclein related diseases called synucleinopathies. In addition, repetitions of and specific mutations to the SNCA gene, the gene that encodes α-syn, results in an increased disposition for synucleinopathies. The latest advances in cryo-EM structure determination and real-space helical reconstruction methods have resulted in over 60 in vitro structures of α-syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 Å. Here, we provide a protocol for α-syn protein expression, purification, and fibrilization. We detail how sample quality is assessed by negative stain transmission electron microscopy (NS-TEM) analysis and followed by sample vitrification using the Vitrobot Mark IV vitrification robot. We provide a detailed step by step protocol for high resolution cryo-EM structure determination of α-syn fibrils using RELION and a series of specialized helical reconstruction tools that can be run within RELION. Finally, we detail how ChimeraX, Coot, and Phenix are used to build and refine a molecular model into the high resolution cryo-EM map. This workflow resulted in a 2.04 Å structure of α-syn fibrils with excellent resolution of residues 36 to 97 and an additional island of density for residues 15 to 22 that had not been previously reported. This workflow should serve as a starting point for individuals new to the neurodegeneration and structural biology fields. Together, this procedure lays the foundation for advanced structural studies of α-synuclein and other amyloid fibrils.Key Features: In vitro fibril amplification method yielding twisting fibrils that span several micrometers in length and are suitable for cryo-EM structure determination. High-throughput cryo-EM data collection of neurodegenerative fibrils, such as alpha-synuclein.Use of RELION implementations of helical reconstruction algorithms to generate high-resolution 3D structures of a-synuclein fibrils.Brief demonstration of the use of ChimeraX, Coot, and Phenix for molecular model building and refinement.s.
Graphical overview of α-synuclein fibrilization and cryo-EM structure determination: α-synuclein protein expression and purification is followed by a fibrilization protocol yielding twisting filaments that span several micrometers in length and are validated by negative stain transmission electron microscopy (NS-TEM). The sample is then vitrified, followed by cryo-EM data collection. Real-space helical reconstruction is performed in RELION to generate an electron potential map that is used for model building.

DOI: https://doi.org/10.1101/2024.09.18.613698
Biology (Basel). 2024 Sep 12. pii: 719. [Epub ahead of print]13(9):

From Brain to Muscle: The Role of Muscle Tissue in Neurodegenerative Disorders.

Elisa Duranti, Chiara Villa.

  Neurodegenerative diseases (NDs), like amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and Parkinson's disease (PD), primarily affect the central nervous system, leading to progressive neuronal loss and motor and cognitive dysfunction. However, recent studies have revealed that muscle tissue also plays a significant role in these diseases. ALS is characterized by severe muscle wasting as a result of motor neuron degeneration, as well as alterations in gene expression, protein aggregation, and oxidative stress. Muscle atrophy and mitochondrial dysfunction are also observed in AD, which may exacerbate cognitive decline due to systemic metabolic dysregulation. PD patients exhibit muscle fiber atrophy, altered muscle composition, and α-synuclein aggregation within muscle cells, contributing to motor symptoms and disease progression. Systemic inflammation and impaired protein degradation pathways are common among these disorders, highlighting muscle tissue as a key player in disease progression. Understanding these muscle-related changes offers potential therapeutic avenues, such as targeting mitochondrial function, reducing inflammation, and promoting muscle regeneration with exercise and pharmacological interventions. This review emphasizes the importance of considering an integrative approach to neurodegenerative disease research, considering both central and peripheral pathological mechanisms, in order to develop more effective treatments and improve patient outcomes.

Keywords:  muscle; neurodegenerative diseases; therapy

DOI:  https://doi.org/10.3390/biology13090719
bioRxiv. 2024 Sep 19. pii: 2024.09.18.613763. [Epub ahead of print]

Alpha-synuclein preformed fibril-induced aggregation and dopaminergic cell death in cathepsin D overexpression and ZKSCAN3 knockout mice.

Toni Mueller, Parker Jeffrey, Yecheng He, Xiaosen Ouyang, David Westbrook, Victor Darley-Usmar, Matthew S Goldberg, Laura Volpicelli-Daley, Jianhua Zhang.

α-synuclein accumulation is recognized as a prominent feature in the majority of Parkinson's disease cases and also occurs in a broad range of neurodegenerative disorders including Alzheimer's disease. It has been shown that α-synuclein can spread from a donor cell to neighboring cells and thus propagate cellular damage, antagonizing the effectiveness of therapies such as transplantation of fetal or iPSC derived dopaminergic cells. As we and others previously have shown, insufficient lysosomal function due to genetic mutations or targeted disruption of cathepsin D can cause α-synuclein accumulation. We here investigated whether overexpression of cathepsin D or knockout (KO) of the transcriptional suppressor of lysosomal biogenesis ZKSCAN3 can attenuate propagation of α-synuclein aggregation and cell death. We examined dopaminergic neurodegeneration in the substantia nigra using stereology of tyrosine hydroxylase-immunoreactive cells 4 months and 6 months after intrastriatal injection of α-synuclein preformed fibrils or monomeric α-synuclein control in control, central nervous system (CNS)-cathepsin D overexpressing and CNS-specific ZKSCAN3 KO mice. We also examined pS129-α-synuclein aggregates in the substantia nigra, cortex, amygdala and striatum. The extent of dopaminergic neurodegeneration and pS129-α-synuclein aggregation in the brains of CNS-specific ZKSCAN3 knockout mice and CNS-cathepsin D overexpressing mice was similar to that observed in wild-type mice. Our results indicate that neither enhancing cathepsin D expression nor disrupting ZKSCAN3 in the CNS is sufficient to attenuate pS129-α-synuclein aggregate accumulation or dopaminergic neurodegeneration.

DOI: https://doi.org/10.1101/2024.09.18.613763
Alzheimers Dement. 2024 Oct 03.

A novel peptide-based tau aggregation inhibitor as a potential therapeutic for Alzheimer's disease and other tauopathies.

Anthony Aggidis, George Devitt, Yongrui Zhang, Shreyasi Chatterjee, David Townsend, Nigel J Fullwood, Eva Ruiz Ortega, Airi Tarutani, Masato Hasegawa, Amber Cooper, Philip Williamson, Ayde Mendoza-Oliva, Marc I Diamond, Amritpal Mudher, David Allsop.

  INTRODUCTION: As aggregation underpins Tau toxicity, aggregation inhibitor peptides may have disease-modifying potential. They are therefore currently being designed and target either the 306VQIVYK311 aggregation-promoting hotspot found in all Tau isoforms or the 275VQIINK280 aggregation-promoting hotspot found in 4R isoforms. However, for any Tau aggregation inhibitor to potentially be clinically relevant for other tauopathies, it should target both hotspots to suppress aggregation of Tau isoforms, be stable, cross the blood-brain barrier, and rescue aggregation-dependent Tau phenotypes in vivo.METHODS: We developed a retro-inverso, stable D-amino peptide, RI-AG03 [Ac-rrrrrrrrGpkyk(ac)iqvGr-NH2], based on the 306VQIVYK311 hotspots which exhibit these disease-relevant attributes.
RESULTS: Unlike other aggregation inhibitors, RI-AG03 effectively suppresses aggregation of multiple Tau species containing both hotspots in vitro and in vivo, is non-toxic, and suppresses aggregation-dependent neurodegenerative and behavioral phenotypes.
DISCUSSION: RI-AG03 therefore meets many clinically relevant requirements for an anti-aggregation Tau therapeutic and should be explored further for its disease-modifying potential for Tauopathies.
HIGHLIGHTS: Our manuscript describes the development of a novel peptide inhibitor of Tau aggregation, a retro-inverso, stable D-amino peptide called RI-AG03 that displays many clinically relevant attributes. We show its efficacy in preventing Tau aggregation in both in vitro and in vivo experimental models while being non-toxic to cells. RI-AG03 also rescues a biosensor cell line that stably expresses Tau repeat domains with the P301S mutation fused to Cer/Clo and rescues aggregation-dependent phenotypes in vivo, suppressing neurodegeneration and extending lifespan. Collectively our data describe several properties and attributes of RI-AG03 that make it a promising disease-modifying candidate to explore for reducing pathogenic Tau aggregation in Tauopathies such as Alzheimer's disease. Given the real interest in reducing Tau aggregation and the potential clinical benefit of using such agents in clinical practice, RI-AG03 should be investigated further for the treatment of Tauopathies after validation in mammalian models. Tau aggregation inhibitors are the obvious first choice as Tau-based therapies as much of Tau-mediated toxicity is aggregation dependent. Indeed, there are many research efforts focusing on this therapeutic strategy with aggregation inhibitors being designed against one of the two aggregation-promoting hotspots of the Tau protein. To our knowledge, RI-AG03 is the only peptide aggregation inhibitor that inhibits aggregation of Tau by targeting both aggregation-promoting hotspot motifs simultaneously. As such, we believe that our study will have a significant impact on drug discovery efforts in this arena.

Keywords:  Alzheimer's disease; Drosophila; VQIINK; VQIVYK; aggregation; amino‐acid; dementia; drug development; in silico; in vitro; in vivo; inhibitor; melanogaster; peptide; tau; tauopathies; therapeutic

DOI:  https://doi.org/10.1002/alz.14246
Acta Neuropathol Commun. 2024 Oct 03. 12(1): 156

Targeting the TDP-43 low complexity domain blocks spreading of pathology in a mouse model of ALS/FTD.

Elodie Chevalier, Mickael Audrain, Monisha Ratnam, Romain Ollier, Aline Fuchs, Kasia Piorkowska, Andrea Pfeifer, Marie Kosco-Vilbois, Tamara Seredenina, Tariq Afroz.

  Abnormal cytoplasmic localization and accumulation of pathological transactive response DNA binding protein of 43 kDa (TDP-43) underlies several devastating diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP). A key element is the correlation between disease progression and spatio-temporal propagation of TDP-43-mediated pathology in the central nervous system. Several lines of evidence support the concept of templated aggregation and cell to cell spreading of pathological TDP-43. To further investigate this mechanism in vivo, we explored the efficacy of capturing and masking the seeding-competent region of extracellular TDP-43 species. For this, we generated a novel monoclonal antibody (mAb), ACI-6677, that targets the pathogenic protease-resistant amyloid core of TDP-43. ACI-6677 has a picomolar binding affinity for TDP-43 and is capable of binding to all C-terminal TDP-43 fragments. In vitro, ACI-6677 inhibited TDP-43 aggregation and boosted removal of pathological TDP-43 aggregates by phagocytosis. When injecting FTLD-TDP brain extracts unilaterally in the CamKIIa-hTDP-43NLSm mouse model, ACI-6677 significantly limited the induction of phosphorylated TDP-43 (pTDP-43) inclusions. Strikingly, on the contralateral side, the mAb significantly prevented pTDP-43 inclusion appearance exemplifying blocking of the spreading process. Taken together, these data demonstrate for the first time that an immunotherapy targeting the protease-resistant amyloid core of TDP-43 has the potential to restrict spreading, substantially slowing or stopping progression of disease.

Keywords:  ALS; FTD; Immunotherapy; Neuropathology; Pathomechanism; Spreading; TDP-43

DOI:  https://doi.org/10.1186/s40478-024-01867-z
Antioxidants (Basel). 2024 Sep 20. pii: 1138. [Epub ahead of print]13(9):

Exploring the Role of Reactive Oxygen Species in the Pathogenesis and Pathophysiology of Alzheimer's and Parkinson's Disease and the Efficacy of Antioxidant Treatment.

Talin Gogna, Benjamin E Housden, Annwyne Houldsworth.

  Alzheimer's (AD) and Parkinson's Disease (PD) are life-altering diseases that are characterised by progressive memory loss and motor dysfunction. The prevalence of AD and PD is predicted to continuously increase. Symptoms of AD and PD are primarily mediated by progressive neuron death and dysfunction in the hippocampus and substantia nigra. Central features that drive neurodegeneration are caspase activation, DNA fragmentation, lipid peroxidation, protein carbonylation, amyloid-β, and/or α-synuclein formation. Reactive oxygen species (ROS) increase these central features. Currently, there are limited therapeutic options targeting these mechanisms. Antioxidants reduce ROS levels by the induction of antioxidant proteins and direct neutralisation of ROS. This review aims to assess the effectiveness of antioxidants in reducing ROS and neurodegeneration. Antioxidants enhance major endogenous defences against ROS including superoxide dismutase, catalase, and glutathione. Direct neutralisation of ROS by antioxidants protects against ROS-induced cytotoxicity. The combination of Indirect and direct protective mechanisms prevents ROS-induced α-synuclein and/or amyloid-β formation. Antioxidants ameliorate ROS-mediated oxidative stress and subsequent deleterious downstream effects that promote apoptosis. As a result, downstream harmful events including neuron death, dysfunction, and protein aggregation are decreased. The protective effects of antioxidants in human models have yet to directly replicate the success seen in cell and animal models. However, the lack of diversity in antioxidants for clinical trials prevents a definitive answer if antioxidants are protective. Taken together, antioxidant treatment is a promising avenue in neurodegenerative disease therapy and subsequent clinical trials are needed to provide a definitive answer on the protective effects of antioxidants. No current treatment strategies have significant impact in treating advanced AD and PD, but new mimetics of endogenous mitochondrial antioxidant enzymes (Avasopasem Manganese, GC4419 AVA) may be a promising innovative option for decelerating neurodegenerative progress in the future at the mitochondrial level of OS.

Keywords:  Alzheimer’s disease; Parkinson’s disease; antioxidants; neurodegeneration; neuroinflammation; oxidative stress; reactive oxygen species

DOI:  https://doi.org/10.3390/antiox13091138
Biophys Rev (Melville). 2024 Sep;5(3): 032104

Capturing the illusive ring-shaped intermediates in Aβ42 amyloid formation.

Yu Yuan, Xiaozhe Dong, Huan Wang, Feng Gai.

Protein/peptide amyloid fibril formation is associated with various neurodegenerative diseases and, hence, has been the subject of extensive studies. From a structure-evolution point of view, we now know a great deal about the initial and final states of this process; however, we know very little about its intermediate states. Herein, we employ liquid-phase transmission electron microscopy to directly visualize the formation of one of the intermediates formed during the aggregation process of an amyloid-forming peptide. As shown in figure, we find that Aβ42, the amyloid formation of which has been linked to the development of Alzheimer's disease, can populate a ring-shaped intermediate structure with a diameter of tens of nanometers; additionally, the air-liquid interface can "catalyze" the formation of amyloid fibrils.

DOI: https://doi.org/10.1063/5.0222349
ACS Chem Neurosci. 2024 Oct 02.

TDP-43 Amyloid Fibril Formation via Phase Separation-Related and -Unrelated Pathways.

Pin-Han Lin, Guan-Wei Wu, Yu-Hao Lin, Jing-Rou Huang, U-Ser Jeng, Wei-Min Liu, Jie-Rong Huang.

  Intrinsically disordered regions (IDRs) in proteins can undergo liquid-liquid phase separation (LLPS) for functional assembly, but this increases the chance of forming disease-associated amyloid fibrils. Not all amyloid fibrils form through LLPS however, and the importance of LLPS relative to other pathways in fibril formation remains unclear. We investigated this question in TDP-43, a motor neuron disease and dementia-causing protein that undergoes LLPS, using thioflavin T (ThT) fluorescence, NMR, transmission electron microscopy (TEM), and wide-angle X-ray scattering (WAXS) experiments. Using a fluorescence probe modified from ThT strategically designed for targeting protein assembly rather than β-sheets and supported by TEM images, we propose that the biphasic ThT signals observed under LLPS-favoring conditions are due to the presence of amorphous aggregates. These aggregates represent an intermediate state that diverges from the direct pathway to β-sheet-dominant fibrils. Under non-LLPS conditions in contrast (at low pH or at physiological conditions in a construct with key LLPS residues removed), the protein forms a hydrogel. Real-time WAXS data, ThT signals, and TEM images collectively demonstrate that the gelation process circumvents LLPS and yet still results in the formation of fibril-like structural networks. We suggest that the IDR of TDP-43 forms disease-causing amyloid fibrils regardless of the formation pathway. Our findings shed light on why both LLPS-promoting and LLPS-inhibiting mutants are found in TDP-43-related diseases.

Keywords:  NMR; TDP-43; amyloid fibril; intrinsically disordered proteins; liquid−liquid phase separation; wide-angle X-ray scattering

DOI:  https://doi.org/10.1021/acschemneuro.4c00503