bims-proned 2025-12-14 papers

bims-proned

Biomed News

on Proteostasis in neurodegeneration

Issue of 2025–12–14
nineteen papers selected by
Verena Kohler, Umeå University

Probing Early α-Synuclein Oligomers: Insights Into Aggregation Pathways of NACore and preNAC Segments Probed by Trapped Ion-Mobility Mass Spectrometry and Fluorescence Spectroscopy.
Human Wild-Type α-Synuclein Drives Progressive Tau Pathology in a Disease-Relevant Mouse Model of Synucleinopathy.
Modulatory Effect of the Polyphenol EGCG toward the Fibrillation of Monomers and Preformed Aggregates of α-Synuclein in the Presence of Small-Molecule and Polymeric Crowders.
Cellular prion protein and calcium ions trigger the neurotoxicity of α-synuclein aggregates.
NEDD8, stress granules, and amyotrophic lateral sclerosis: unveiling the therapeutic potential of the NEDP1 protease.
Human RBM3 protein is prone to form neuronal aggregates opposed by the proteasome.
Ubiquitin Proteasome System Components, RAD23A and USP13, Modulate TDP-43 Solubility and Neuronal Toxicity.
AI-Guided Dual Strategy for Peptide Inhibitor Design Targeting Structural Polymorphs of α-Synuclein Fibrils.
Heparin and fluoride drive distinct tau (4R/1 N) aggregation pathways to fibrils and granular oligomers, as revealed by Raman spectroscopy.
Decoding the role of large heat shock proteins in the progression of neuroinflammation-mediated neurodegenerative disorders.
A Dual-Targeted Fluorescent Probe with a Semicurcumin Structure for Imaging β-Amyloid and α-Synuclein Aggregates.
Skin-derived α-synuclein strains from PD, DLB, and MSA induce distinct intracellular pathology and neurodegeneration.
Amyloid-motif-dependent tau self-assembly is modulated by isoform sequence context.
N-terminal KTKEGV motif lysine residues of α-Synuclein are critical for TLR2 interaction and activation.
Nitrated products formed on α-synuclein are preferentially incorporated into oligomers but excluded from fibrils: A mechanism for accumulation of neurotoxic species.
Study of the Aggregation Behavior of Proteins Related to Neurodegenerative Diseases Based on the Photoluminescence of Perovskite Nanocrystals.
APOE isoform-associated tau oligomer polymorphs differ in synaptotoxicity and seeding activity.
PolyQ Expansion Controls Biomolecular Condensation and Aggregation of the N-Terminal Fragments of Ataxin-2.
A One-Step Mouse Model of Parkinson's Disease Combining rAAV-α-Synuclein and Preformed Fibrils of α-Synuclein.

Proteomics. 2025 Dec 10. e70087

Probing Early α-Synuclein Oligomers: Insights Into Aggregation Pathways of NACore and preNAC Segments Probed by Trapped Ion-Mobility Mass Spectrometry and Fluorescence Spectroscopy.

Agathe Depraz Depland, Stephanie Mikromanolis, Iuliia Stroganova, Anouk M Rijs.

  Misfolding and aggregation of α-Synuclein (α-Syn) play a central role in Parkinson's disease (PD), with oligomeric intermediates implicated as key toxic species. Here, we investigate the aggregation of two α-Syn segments, the NACore (68GAVVTGVTAVA78, WT-PD1) and the preNAC region (47GVVHGVATVA56, WT-PD2), using high-resolution trapped ion-mobility mass spectrometry (TIMS-Qq-ToF) and Thioflavin T fluorescence spectroscopy. The NACore is the minimal sequence required for α-Syn aggregation, whereas most mutations affecting the onset of PD appear to be in the preNAC region, therefore modulating aggregation dynamics and toxicity. Our results demonstrate that TIMS-Qq-ToF effectively resolves and identifies oligomeric species, revealing distinct aggregation pathways for both peptide segments. Fluorescence assays confirm differences in aggregation kinetics and morphology, highlighting the different oligomer formation pathways observed with ion-mobility mass spectrometry. Despite the oligomer-preserving nature of the TIMS itself, ion transmission remains too harsh for the fragile oligomers, leading to fragmentation of fragile non-covalent assemblies. However, collisional cross-section (N2CCSTIMS) values support the presence of large oligomers. This study highlights the potential of TIMS-Qq-ToF for mapping α-Syn aggregation and underscores the need for optimised, soft ion transmission to better preserve fragile transient intermediates, ultimately contributing to a deeper understanding of PD. SUMMARY: Understanding early-stage protein aggregation is essential for unravelling the molecular mechanisms of Parkinson's disease, a neurodegenerative disorder that currently lacks effective therapeutic solutions. This study employs high-resolution Trapped Ion Mobility Mass Spectrometry combined with fluorescence spectroscopy to elucidate the oligomerisation of two α-synuclein segments, revealing distinct aggregation pathways and associated structural characteristics. This study underscores the value of peptide models in advancing our understanding of protein aggregation behaviour. This multifaceted approach provides detailed structural insights into the unexplored, transient early-stage oligomers of key α-Syn segments, contributing to a deeper understanding of aggregation mechanisms and providing valuable insights for therapeutic strategies against Parkinson's disease.

Keywords:  NACore; Parkinson's disease; ThT fluorescence; early‐stage oligomers; peptide aggregation; trapped ion‐mobility mass spectrometry

DOI:  https://doi.org/10.1002/pmic.70087
Res Sq. 2025 Dec 03. pii: rs.3.rs-8166445. [Epub ahead of print]

Human Wild-Type α-Synuclein Drives Progressive Tau Pathology in a Disease-Relevant Mouse Model of Synucleinopathy.

Sudipta Senapati, Alessia Sciortino, Madison Samples, Nikita Shchankin, Yingxin Zhao, Mauro Montalbano, Rakez Kayed.

Tau pathology, characterized by the aberrant aggregation and accumulation of tau in neurons and glial cells, plays a critical role in the onset and progression of multiple neurodegenerative diseases. Although synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are primarily defined by intracytoplasmic inclusions of α-Synuclein (α-Syn), they frequently exhibit substantial tau co-pathology. Emerging genetic and pathological evidence suggests a mechanistic interplay between α-Syn and tau that amplifies their aggregation and accelerates neurodegeneration. In this study, we systematically investigated the temporal progression of tau pathology in a transgenic PD mouse model that overexpresses human wild-type (WT) α-Syn (hSyn mice). Prior work has shown that this model develops α-Syn pathology across key brain regions, including the substantia nigra, cortex, hippocampus, and striatum, accompanied by microglial activation and synaptic dysfunction. Using a combination of biochemical, biophysical, and immunological approaches, we demonstrate a progressive accumulation of hyperphosphorylated tau, as well as soluble and insoluble tau aggregates, in the brains of hSyn mice. Electron microscopy of insoluble fractions reveals abundant fibrillar structures, while mass spectrometry confirms that these fibrils consist of both α-Syn and tau. Notably, these pathologies are absent in WT littermates, suggesting that tau aggregation arises as a consequence of α-Syn overexpression rather than normal aging. Collectively, our findings establish a mechanistic link between α-Syn and tau aggregation, identifying tau as an active contributor to α-Syn-driven neurodegeneration. This study provides direct experimental evidence that tau co-pathology contributes to disease progression in synucleinopathies, underscoring the therapeutic potential of targeting pathological tau to mitigate neurodegeneration in PD, DLB, and related disorders.

DOI: https://doi.org/10.21203/rs.3.rs-8166445/v1
J Phys Chem B. 2025 Dec 11.

Modulatory Effect of the Polyphenol EGCG toward the Fibrillation of Monomers and Preformed Aggregates of α-Synuclein in the Presence of Small-Molecule and Polymeric Crowders.

Santosh Devi, Rajiv Bhat.

Epigallocatechin gallate (EGCG) has been widely studied for its inhibitory effects on amyloidogenic protein aggregation, yet its effect on the intrinsically disordered neuronal protein α-synuclein (α-syn), which is implicated in many neurodegenerative disorders, remains unclear, especially under molecular crowding conditions that mimic cellular environments. However, recent studies have reported contrasting effects of EGCG on α-syn aggregation. This study comprehensively examines the effect of EGCG on monomeric α-syn aggregation and preformed aggregates in the absence and presence of small (polyol osmolytes) and large (PEG-8000) molecular crowders. Our results show that EGCG delays the lag phase of α-syn aggregation in the presence of polyols, whereas no significant change is observed in the presence of the large molecular weight crowder PEG8K. Atomic force microscopy images reveal that EGCG distinctly redirects α-syn to form oligomers and amorphous aggregates under crowded conditions. Furthermore, while α-syn, in the presence of a combination of EGCG and PEG8K, exhibits random coil and α-helical conformations, it shows a completely disordered conformation in its absence. Interestingly, the addition of EGCG to preformed aggregates does not alter aggregate size but impacts their morphology significantly, as shown by light scattering and AFM. Despite the changes in the morphology of aggregates, a predominantly β-sheet-rich secondary structure was observed by CD spectroscopy after EGCG addition, suggesting complexity in the action of EGCG under crowding conditions. Our study highlights the critical role played by the physicochemical nature of the cellular environment, consisting of small-molecule osmolytes as well as macromolecular crowders, in protein aggregation and its modulation by small-molecule ligands such as EGCG.

DOI: https://doi.org/10.1021/acs.jpcb.5c05714
Cell Biosci. 2025 Dec 09. 15(1): 166

Cellular prion protein and calcium ions trigger the neurotoxicity of α-synuclein aggregates.

Alessandra Bigi, Andrea Carlotta Conti, Liliana Napolitano, Giuliana Fusco, Alfonso De Simone, Fabrizio Chiti, Roberta Cascella, Cristina Cecchi.

   BACKGROUND: The aggregation of α-Synuclein (αS) into amyloid fibrils and their deposition in intraneuronal Lewy bodies are hallmark features of Parkinson's disease (PD) and other synucleinopathies. Among the molecular players implicated in αS toxicity, the cellular prion protein (PrPC) has emerged as a potential modulator of αS-neuron interactions.
RESULTS: Using confocal microscopy, colocalization analysis and both siRNA-induced PrPC silencing and antibody-based blockade, we investigated the contribution of PrPC to αS-induced neurotoxicity in human iPSC-derived dopaminergic neurons, primary rat cortical neurons and human SH-SY5Y neuroblastoma cells. We show that PrPC facilitated the early recruitment of αS prefibrillar type B* oligomers (OB*) and short fibrils (SF) to neuronal membranes, enhancing αS-induced Ca2+ influx and membrane permeabilization. However, PrPC levels remained unchanged following prolonged exposure with OB* and SF, suggesting no feedback modulation of PrPC expression. While PrPC blockade partially inhibited the release of toxic soluble oligomers from αS fibrils, downstream cell death was only marginally reduced, indicating a limited contribution of PrPC to the final neurotoxic outcome. By contrast, extracellular Ca2+ emerged as a major driver of αS toxicity, directly promoting the membrane recruitment, internalization and cytotoxic effects of αS aggregates.
CONCLUSIONS: Collectively, our findings indicate that while PrPC facilitates early events in αS aggregate interaction with neurons, the sustained neurotoxicity induced by αS prefibrillar oligomers and fibrils is predominantly mediated by extracellular Ca2+. This promotes aggregate-membrane interactions, membrane permeabilization, and intracellular Ca2+ dyshomeostasis, thereby establishing a vicious cycle of neuronal dysfunction and death.

Keywords:  Amyloid; Fibrils; Lewy bodies; Neurodegeneration; Parkinson's Disease; Protein aggregation; Protein misfolding; Synucleinopathies; Toxic oligomers

DOI:  https://doi.org/10.1186/s13578-025-01479-7
Essays Biochem. 2025 Dec 12. pii: EBC20253036. [Epub ahead of print]

NEDD8, stress granules, and amyotrophic lateral sclerosis: unveiling the therapeutic potential of the NEDP1 protease.

Dimitra Mitsiadou, Dimitris P Xirodimas, Jolanta Polanowska.

  Protein quality control (PQC) systems are crucial for maintaining cellular proteostasis, particularly under stress that promotes misfolded protein accumulation. A central component of this response is the assembly of stress granules (SGs), cytoplasmic condensates of RNA and proteins that temporarily stall translation. Aberrant SG dynamics, often linked to mutations in SG proteins, contribute to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), where persistent protein aggregates are hallmarks. This review examines the emerging role of the ubiquitin-like modifier NEDD8 and its deconjugating enzyme NEDP1 in regulating SG homeostasis. Recent studies identify NEDP1 as a critical factor controlling SG clearance. Inhibition of NEDP1 enhances SG turnover, prevents pathological solidification, and promotes the disassembly of toxic aggregates through hyper-NEDDylation of PARP1, a DNA repair enzyme that also governs SG dynamics. Unlike broad-spectrum PARP1 inhibitors, which can impair DNA repair and cause cytotoxicity, NEDP1 inhibition offers a stress-specific approach that preserves normal cellular functions. Encouragingly, NEDP1 inhibition effectively causes aggregate elimination in ALS patient-derived fibroblasts and restores motility in Caenorhabditis elegans disease models. Altogether, these findings highlight NEDP1 as a key regulator of SG regulation and a promising therapeutic target for ALS and related neurodegenerative disorders.

Keywords:  NEDD8; NEDP1/SENP8; PARP1; amyotrophic lateral sclerosis; stress granules

DOI:  https://doi.org/10.1042/EBC20253036
Biol Open. 2025 Dec 12. pii: bio.062179. [Epub ahead of print]

Human RBM3 protein is prone to form neuronal aggregates opposed by the proteasome.

Suman Kumar, Tina Kleven, Rafal Ciosk.

  Maintenance of proteostasis is critical for neuronal functions, as the accumulation of misfolded or damaged proteins leads to neurodegeneration. Cooling is generally neuroprotective and is used in various clinical settings. However, how it impacts neuronal proteostasis remains unclear. In rodents, the neuroprotective effects of cold have been largely attributed to the cold-inducible protein RBM3. Here, studying the human RBM3 in cultured neurons subjected to profound hypothermia, we observed its cold-induced aggregation. These RBM3 aggregates are distinct from stress granules, occur specifically in differentiated neurons, and form also at physiological temperature upon proteasomal inhibition. Thus, in humans, RBM3 aggregation may be normally counteracted by the proteasome to maintain neuronal health. Exploring the natural variation between RBM3 proteins in hibernating versus non-hibernating mammals, we discuss how the aggregation could be prevented in animals with fluctuating body temperature. These findings are important for the understanding of RBM3 functions and neuronal proteostasis and have implications for medical treatments involving incidental and induced hypothermia.

Keywords:  Aggregation; Hibernation; Hypothermia; Neurodegeneration; Proteostasis; RBM3

DOI:  https://doi.org/10.1242/bio.062179
J Neurosci. 2025 Dec 10. pii: e0906252025. [Epub ahead of print]

Ubiquitin Proteasome System Components, RAD23A and USP13, Modulate TDP-43 Solubility and Neuronal Toxicity.

Casey Dalton, Jelena Mojsilovic-Petrovic, Nathaniel Safren, Carley Snoznik, Kamil K Gebis, Yi-Zhi Wang, Alexandra B Sutter, Todd Lamitina, Jeffrey N Savas, Robert G Kalb.

At autopsy, >95% of ALS cases display a redistribution of the essential RNA binding protein TDP-43 from the nucleus into cytoplasmic aggregates. The mislocalization and aggregation of TDP-43 is believed to be a key pathological driver in ALS. Due to its vital role in basic cellular mechanisms, direct depletion of TDP-43 is unlikely to lead to a promising therapy. Therefore, we have explored the utility of identifying genes that modify its mislocalization or aggregation. We have previously shown that loss of rad-23 improves locomotor deficits in TDP-43 C. elegans models of disease and increases the degradation rate of TDP-43 in cellular models. To understand the mechanism through which these protective effects occur, we generated an inducible mutant TDP-43 HEK293 cell line. We find that knockdown of RAD23A reduces insoluble TDP-43 levels in this model and primary rat cortical neurons expressing human TDP-43A315T Utilizing a discovery-based proteomics approach, we then explored how loss of RAD23A remodels the proteome. Through this proteomic screen, we identified USP13, a deubiquitinase, as a new potent modifier of TDP-43 induced aggregation and cytotoxicity. We find that knockdown of USP13 reduces the abundance of sarkosyl insoluble mTDP-43 in both our HEK293 model and primary rat neurons, reduces cell death in primary rat motor neurons, and improves locomotor deficits in C. elegans ALS models.Significance Statement Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease (NDD) with no effective therapies. The mislocalization and aggregation of TAR DNA binding protein 43 (TDP-43) is a key pathological marker of ALS and other NDDs. Due to its vital functions, targeted therapeutic reduction of TDP-43 could be problematic. Here, we have explored the utility of targeting modifier genes. We find that knockdown of two members of the ubiquitin proteasome system, RAD23A and USP13, enhance TDP-43 solubility and decrease TDP-43 induced neurotoxicity.

DOI: https://doi.org/10.1523/JNEUROSCI.0906-25.2025
Cells. 2025 Dec 03. pii: 1921. [Epub ahead of print]14(23):

AI-Guided Dual Strategy for Peptide Inhibitor Design Targeting Structural Polymorphs of α-Synuclein Fibrils.

Jinfang Duan, Haoyu Zhang, Chuanqi Sun.

  One of the most important events in the pathogenesis of Parkinson's disease and related disorders is the formation of abnormal fibrils via the aggregation of α-synuclein (α-syn) with β-sheet-rich organization. The use of Cryo-EM has uncovered different polymorphs of the fibrils, each having unique structural interfaces, which has made the design of inhibitors even more challenging. Here, a structure-guided framework incorporating AI-assisted peptide generation was set up with the objective of targeting the conserved β-sheet motifs that are present in various forms of α-syn fibrils. The ProteinMPNN, then, AlphaFold-Multimer, and PepMLM were employed to create short peptides that would interfere with the growth of the fibrils. The two selected candidates, T1 and S1, showed a significant inhibition of α-syn fibrillation, as measured by a decrease in the ThT fluorescence and the generation of either amorphous or fragmented aggregates. The inhibitory potency of the peptides was in line with the predicted interface energies. This research work illustrates that the integration of cryo-EM structural knowledge with the computational design method leads to the quick discovery of the wide-spectrum peptide inhibitors, which is a good strategy for the precision treatment of neurodegenerative diseases.

Keywords:  Parkinson’s disease; artificial intelligence; cryo-electron microscopy; peptide inhibitor design; structural polymorphism; α-synuclein fibrils

DOI:  https://doi.org/10.3390/cells14231921
J Biomol Struct Dyn. 2025 Dec 11. 1-17

Heparin and fluoride drive distinct tau (4R/1 N) aggregation pathways to fibrils and granular oligomers, as revealed by Raman spectroscopy.

Hadi Rouhbakhsh, Nahid Farkhari, Seyyed Hossein Khatami, Sajad Ehtiati, Mohsen Masoumian Hosseini, Yaser Soleimani, Mohammad-Reza Rashidi, Habib Tajalli, Sohrab Ahmadi-Kandjani, Amir Boostanipour, Amir Mohammad Karimi, Saeed Karima.

  The aggregation of the tau protein into pathological assemblies is a pivotal event in Alzheimer's disease and related tauopathies. Understanding how different cofactors influence the Tau aggregation pathway is crucial for elucidating disease mechanisms. This study directly compares the aggregation pathways of human Tau (hTau441, 4 R/1 N) induced by heparin with those induced by aluminum/sodium fluoride (AlF3/NaF). We employed time-resolved Raman spectroscopy, a technique uniquely suited for label-free, real-time secondary structure analysis of intrinsically disordered proteins in solution, to monitor structural transitions. Our results reveal two distinct trajectories: Heparin drives a classical pathway of progressive β-sheet enrichment, culminating in mature fibrils. In stark contrast, fluoride conditions suppress β-sheet formation and stabilize granular, nonfibrillar oligomers. These findings suggest that the neurotoxicity associated with fluoride may not arise from accelerating fibril formation but from diverting tau into an off-pathway oligomeric state. This work establishes Raman spectroscopy as a powerful tool for mechanistic studies of protein aggregation and identifies fluoride as a modulator of Tau misfolding with significant pathological implications.

Keywords:  Raman spectroscopy; Tau aggregation; amide I deconvolution; fluoride-modulated oligomers; heparin-induced fibrils

DOI:  https://doi.org/10.1080/07391102.2025.2598636
Neuroprotection. 2025 Mar;3(1): 48-62

Decoding the role of large heat shock proteins in the progression of neuroinflammation-mediated neurodegenerative disorders.

Shailendra K Saxena, Deepak Sharma, Swatantra Kumar, Vimal K Maurya, Saniya Ansari, Hardeep S Malhotra, Amit Singh.

  Chronic neuroinflammation and protein aggregation are the fundamental events mainly responsible for the progression of neurodegenerative diseases (NDs). Potential neurotoxic changes in the intra- and extracellular environment are typical hallmarks of many NDs. Treatment of ND is challenging, as the symptoms in these patients arises when a significant numbers of neurons have already been destroyed. Heat shock proteins (HSPs) can bind to recipient cells that are susceptible to stress, such as neurons, in the extracellular environment, therefore enhancing stress resistance. Among all, HSP60, HSP70, and HSP90 are highly conserved molecular chaperones involved in protein folding and assembly, maintaining cellular homeostasis in the central nervous system. Notably, α-synuclein accumulation is a major pathophysiology in Parkinson's disease, where HSP90 modulates the assembly of α-synuclein in vesicles to prevent its accumulation. Moreover, HSP90 regulates the activity of the glycogen synthase kinase-3β protein, which is crucial in diabetes mellitus-associated neurocognitive disorder. Therefore, understanding the molecular mechanism by which HSPs facilitate protein aggregation and respond to inflammatory stimuli, including metabolic disease such as diabetes, is essential for understanding the significance of HSPs in NDs. This review emphasizes the role of various HSPs in the progression of NDs such as Alzheimer's, Parkinson's, multiple sclerosis, and Huntington's disease, including diabetes, which is one of the major risk factors for neurodegeneration.

Keywords:  Alzheimer's disease; Huntington's disease; Parkinson's disease; heat shock protein; neurodegenerative disease; neuroinflammation

DOI:  https://doi.org/10.1002/nep3.68
Bioconjug Chem. 2025 Dec 11.

A Dual-Targeted Fluorescent Probe with a Semicurcumin Structure for Imaging β-Amyloid and α-Synuclein Aggregates.

Zeying Liang, Tiantian Tang, Jianyu Wu, Haijun Yang, Yi Zou, Jiapei Dai, Xingdang Liu, Jian Yang.

Alzheimer's disease (AD) and Parkinson's disease (PD) are two of the most prevalent neurodegenerative disorders, both characterized by abnormal protein folding. Amyloid-β (Aβ) and α-synuclein (α-syn) are often associated with these diseases, which might occur simultaneously in later stages. In this study, we developed a dual-target fluorescent probe, DiFboron-6, designed to specifically target Aβ and α-syn aggregates. DiFboron-6 exhibited excellent fluorescence properties, with 59-fold and 49-fold increases in fluorescence intensity upon binding to Aβ and α-syn aggregates, respectively. The probe demonstrated strong binding affinity to both proteins, with dissociation constants of KdAβ = 12.4 nM and Kdα-syn = 174 nM. Mouse brain slice staining and in vitro experiments further confirmed that DiFboron-6 could clearly label both protein plaques and effectively cross the blood-brain barrier (BBB). DiFboron-6 has been shown to effectively detect both Aβ and α-syn aggregates, thereby serving as a dual-target detection tool. Its unique structure offers a promising foundation for the development of future dual-target probes.

DOI: https://doi.org/10.1021/acs.bioconjchem.5c00454
J Biol Chem. 2025 Dec 08. pii: S0021-9258(25)02857-1. [Epub ahead of print] 111005

Skin-derived α-synuclein strains from PD, DLB, and MSA induce distinct intracellular pathology and neurodegeneration.

Anupam Raina, Wen Wang, Jose Carlos Gonzalez, Xiaohui Yan, Linda Overstreet-Wadiche, Jacques I Wadiche, Chun-Li Zhang, Shu G Chen.

  α-Synuclein (αSyn) aggregates ("strains") can be detected by seed amplification assays such as real-time quaking-induced conversion (RT-QuIC) from the skin of patients with synucleinopathies including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). However, whether skin-derived αSyn strains induce disease-specific pathology in a biological system is unknown. We have identified a human glioblastoma cell line U251 that readily forms intracellular αSyn inclusions upon seeding by exogenous αSyn seeds. These intracellular αSyn inclusions are detergent-insoluble and colocalize with phosphorylated-αSyn at serine 129 (p-αSyn), the pathological hallmark of synucleinopathies. We have engineered a FRET-based αSyn biosensor in U251 cells to characterize intracellular aggregation of αSyn and morphology of p-αSyn inclusions seeded by RT-QuIC-amplified patient skin αSyn strains. The skin-derived αSyn strains from PD, DLB, and MSA patients are capable of inducing intracellular αSyn aggregation characterized by FRET-positive inclusions colocalized with p-αSyn. Interestingly, PD skin-amplified strains are more bioactive, which induce a greater pathological burden and a distinct p-αSyn inclusion morphology from DLB skin-amplified strains. Furthermore, the skin-amplified αSyn strains induce neuronal inclusions and trigger degeneration of induced neurons reprogrammed from U251 biosensor cells. Finally, biosensor cell-propagated PD skin αSyn strains induce higher in vitro seeding activity than DLB skin αSyn strains, indicating a strain-specific relationship between intracellular pathological αSyn burden and in vitro seeding activity. In conclusion, αSyn strains derived from PD, DLB, and MSA patient skin are bioactive, pathologically distinct, and trigger neurodegeneration. Our findings emphasize the importance of studying tissue- and strain-specific pathogenesis of synucleinopathies.

Keywords:  Alpha-synuclein; Biosensor cells; Neurodegenerative diseases; RT-QuIC; Skin; Strains

DOI:  https://doi.org/10.1016/j.jbc.2025.111005
Structure. 2025 Dec 05. pii: S0969-2126(25)00443-5. [Epub ahead of print]

Amyloid-motif-dependent tau self-assembly is modulated by isoform sequence context.

Sofia Bali, Pawel M Wydorski, Ruhar Singh, Nico E Van Nuland, Aleksandra Wosztyl, Nabil Morgan, Valerie A Perez, Dailu Chen, Jie Chen, Josep Rizo, Lukasz A Joachimiak.

  The microtubule-associated protein tau is implicated in neurodegenerative diseases characterized by amyloid formation. Mutations associated with frontotemporal dementia increase tau aggregation propensity and disrupt its endogenous microtubule-binding activity. However, the structural relationship between aggregation propensity and biological activity remains unclear. We employed a multi-disciplinary approach, including computational modeling, NMR, cross-linking mass spectrometry, and cell models to engineer tau sequences that modulate its structural ensemble. Our findings show that substitutions near the conserved "PGGG" β-turn motif informed by tau isoform context reduce tau aggregation in vitro and can counteract aggregation from disease-associated proline-to-serine mutations. Engineered tau sequences maintain microtubule binding and explain why 3R isoforms exhibit reduced pathogenesis compared to 4R. We propose a simple mechanism to reduce the formation of pathogenic tau species while preserving biological function, thus offering insights for therapeutic strategies aimed at reducing tau protein misfolding in neurodegenerative diseases.

Keywords:  aggregation; amyloid; amyloid motif; microtubule-associated protein tau; misfolding; protein design; tauopathies

DOI:  https://doi.org/10.1016/j.str.2025.11.009
Arch Biochem Biophys. 2025 Dec 10. pii: S0003-9861(25)00415-1. [Epub ahead of print] 110701

N-terminal KTKEGV motif lysine residues of α-Synuclein are critical for TLR2 interaction and activation.

Krishna Singh Bisht, Manisha Kumari, Tushar Kanti Maiti.

  Synucleinopathies, such as Parkinson's Disease, involve widespread intracellular protein aggregates. α-synuclein (αSyn) protein contributes majorly to these aggregates called as Lewy Bodies, which are linked to neurodegeneration. αSyn has been shown to transmit from infected to healthy neurons, thus spreading the pathology. During its transmission, αSyn is also known to activate microglia by interacting with the glial surface receptors such as Toll-Like receptors (TLRs). This activation is implicated in the production of interleukins and proinflammatory cytokines, exacerbating neuronal death through chronic neuroinflammation. However, the mechanism of αSyn and TLR interaction is not well elucidated. In this study, using biophysical methods and in the HEK-TLR2 cells, we demonstrate that αSyn N-terminal has a higher binding affinity with TLR2. This interaction is mediated through the lysine residues in the second and third KTKEGV motifs of αSyn. The lysine to alanine mutations in the N-terminal KTKGEV motifs perturb the αSyn/TLR2 interaction. We also demonstrate that the lysine residues of 2nd and 3rd KTKEGV motifs are critical for αSyn-mediated TLR2 activation. Our study demonstrates that the N-terminal KTKEGV lysine residues facilitate interaction with TLR2, thereby regulating αSyn-mediated neuroinflammation.

Keywords:  Alpha-Synuclein; Amyloids; Lewy Bodies; Neuroinflammation; Parkison’s Disease; Protein aggregation; Synucleinopathies; TLR2

DOI:  https://doi.org/10.1016/j.abb.2025.110701
Biochim Biophys Acta Proteins Proteom. 2025 Dec 10. pii: S1570-9639(25)00056-1. [Epub ahead of print] 141118

Nitrated products formed on α-synuclein are preferentially incorporated into oligomers but excluded from fibrils: A mechanism for accumulation of neurotoxic species.

Daniel E Otzen, Luke F Gamon, Per Hägglund, Janni Nielsen, Jannik N Pedersen, Tina Nybo, Jan S Nowak, Søren K Amstrup, Mitra Pirhaghi, Michael J Davies.

  Post-translational modifications (PTMs) such as nitration of Tyr (Y) residues and di-tyrosine (DT) formation are known to impact the aggregation behavior of α-synuclein (α-syn), a protein closely linked to Parkinson's disease. Using tetranitromethane (TNM) as a model nitrating agent, we systematically investigated the chemical modifications of α-syn and their consequences for aggregation. Mass spectrometry analysis revealed site-selective nitration of all four Tyr residues, with Y39 and Y125 being most susceptible. DT crosslinks were also observed, primarily involving Y39, but were disfavored at higher TNM concentrations, indicating competition between nitration and crosslinking pathways. Higher TNM concentrations favored nitration over crosslinking, consistent with common radical intermediates. Even sub-stoichiometric amounts of TNM-modified α-syn significantly inhibited fibril elongation, suggesting that nitration disrupts the templated addition of monomers into the ordered fibrillar structure. Consistent with this, TNM-modified α-syn was strongly excluded from fibrillar assemblies. In contrast, they were preferentially incorporated into soluble oligomers, underlining the promiscuous ability of oligomers to act as a sink for chemically modified α-syn monomers, with potential implications for neurotoxicity.

Keywords:  3-nitrotyrosine; Crosslinks; Fibril formation; Neurodegeneration; Parkinson's disease; Tetranitromethane; di-tyrosine

DOI:  https://doi.org/10.1016/j.bbapap.2025.141118
Anal Chem. 2025 Dec 08.

Study of the Aggregation Behavior of Proteins Related to Neurodegenerative Diseases Based on the Photoluminescence of Perovskite Nanocrystals.

Yizhu Wang, Mengxiao Lv, Wenxiu Zhu, Jingyu Yan, Jiaqi Shi, Ziyun Qin, Xiye Zhang, Huiqin Yao, Lingchen Meng, Hongyun Liu, Lanqun Mao.

Neurodegenerative diseases (NDs) are closely associated with abnormal protein aggregation. In this study, SiO2-coated CsPbBr3 nanocrystals (CPB NCs) were exploited as label-free photoluminescence (PL) probes to investigate the aggregation behavior of amyloid β (Aβ), a key protein in Alzheimer's disease. The PL intensity of the CPB NCs in both aqueous dispersion and thin-film states decreased with increasing degree of Aβ aggregation. The relative PL intensity could quantitatively distinguish the aggregation states of Aβ, and the sensitivity of the thin-film system was twice that of the aqueous dispersion. This approach innovatively enabled the preliminary monitoring of protein aggregates without interfering with the aggregation process while avoiding the effects of reactive oxygen species generated during the aggregation and metal ions themselves on PL signals. Through threshold segmentation, the relative fluorescence intensity was further used to distinguish small-molecule drugs with disaggregation effects (ratio > 0.5) from those without (ratio ≈ 0). Based on a series of experiments, the photoinduced electron transfer mechanism was proposed to explain the change in the PL signal. Due to the electrostatic interaction between CPB NCs and proteins, this probe had potential for studying the aggregation states of other proteins (such as α-synuclein) related to NDs. Overall, this work offered new insights into the application of metal halide perovskites in biosensing as well as for the diagnosis and drug screening of NDs.

DOI: https://doi.org/10.1021/acs.analchem.5c05070
Alzheimers Dement. 2025 Dec;21(12): e70965

APOE isoform-associated tau oligomer polymorphs differ in synaptotoxicity and seeding activity.

Naomi Moreno, Nikita Shchankin, Leiana Fung, Nicha Puangmalai, Nemil Bhatt, Md Anzarul Haque, Yingxin Zhao, C Dirk Keene, Agenor Limon, Rakez Kayed.

   INTRODUCTION: Pathological tau aggregates form distinct polymorphic species across diseases and even across Alzheimer's disease (AD) patients. However, tau aggregate polymorphism across the apolipoprotein E isoforms (APOE ε2, ε3, ε4), the strongest predictors of late-onset AD development, is unknown.
METHODS: This study assessed the conformational and bioactivity properties of tau oligomers from 14 patients with varying APOE genotypes.
RESULTS: Tau oligomers differ in proteolytic stability and cleavage site profiles across the APOE isoforms, indicating conformationally distinct polymorphs. APOE isoform-associated tau oligomers affect synaptic plasticity differently, with ε4-associated oligomers having the highest potency and strongest impact on synaptic functioning. Bioactivity assays reveal that ε4-associated oligomers demonstrate particularly high seeding activity. Interestingly, tau oligomer synaptotoxicity and seeding activity are independent characteristics.
DISCUSSION: The APOE isoforms are associated with distinct tau oligomer polymorphs with varying bioactivity, underscoring the importance of considering APOE status when generating AD therapies. Polymorph-specific targeting of pathological tau species could provide a novel method of combating AD.
HIGHLIGHTS: Conformational and bioactivity distinctions of tau oligomers have not yet been investigated across the APOE isoforms (ε2, ε3, ε4). Tau oligomers differ in conformational properties across the APOE isoforms. APOE ε4-relevant tau oligomers strongly impair synaptic plasticity and demonstrate high tau seeding activity. APOE ε4-relevant tau oligomers exist as a particularly toxic species, making them an ideal target for tau-based AD therapies.

Keywords:  aggregation; oligomers; polymorphs; synaptic functioning; synaptotoxicity; tau

DOI:  https://doi.org/10.1002/alz.70965
Int J Mol Sci. 2025 Nov 28. pii: 11538. [Epub ahead of print]26(23):

PolyQ Expansion Controls Biomolecular Condensation and Aggregation of the N-Terminal Fragments of Ataxin-2.

Yin-Hu Liu, Heng-Tong Duan, Lei-Lei Jiang, Hong-Yu Hu.

  Ataxin-2 (Atx2) is a general RNA-binding protein involved in processes such as RNA processing and metabolism in cells. Atx2 is also a polyglutamine (polyQ) tract-containing protein; its abnormal expansion can lead to protein aggregation associated with neurodegenerative diseases. Previous studies have shown that the C-terminal intrinsically disordered regions (c-IDRs) of Atx2 participate in its condensation and aggregation processes. To elucidate the role of polyQ expansion in biomolecular condensation and aggregation, we studied the N-terminal fragments of Atx2 (namely, Atx2-N317 and Atx2-N81) that preserve a polyQ tract and compared their molecular behaviors in cells to those of the full-length Atx2. We found that the molecular mobility of the N-terminal fragments decreases with the increasing length of polyQ, indicating that polyQ expansion promotes a gradual phase transition to an irreversible and insoluble state. Moreover, the molecular state and mobility of Atx2-N317 are not distinct from those of Atx2-N81, regardless of the presence of other domains, demonstrating that the polyQ tract is a direct and sufficient element for protein condensation and aggregation, while the Like Sm (LSm) and LSm-associated (LSmAD) domains and their interactions with RNA are not necessary for these processes. This result is also validated through the in vitro investigation of Atx2-N81 with different polyQ expansions. This study reveals that polyQ expansion controls the biomolecular condensation and aggregation of the N-terminal fragments of Atx2 and is thus thought to modulate the dynamic behaviors of the full-length protein as well, which is implicated in the pathological accumulation of Atx2 in cells.

Keywords:  aggregation; ataxin-2; biomolecular condensation; molecular dynamics; polyQ expansion

DOI:  https://doi.org/10.3390/ijms262311538
Bio Protoc. 2025 Dec 05. 15(23): e5525

A One-Step Mouse Model of Parkinson's Disease Combining rAAV-α-Synuclein and Preformed Fibrils of α-Synuclein.

Santhosh Kumar Subramanya, Devendra Singh, Poonam Thakur.

  Developing preclinical animal models that faithfully mimic the progressive nature of Parkinson's disease (PD) is crucial for advancing mechanistic insights as well as therapeutic discovery. While recombinant adeno-associated virus (rAAV)-driven α-synuclein overexpression is widely used, its reliance on high viral titers introduces nonspecific toxicity and limits physiological relevance. The SynFib model, which combines modest rAAV-driven α-synuclein expression (Syn) with α-synuclein preformed fibril (PFF) seeding (Fib), has shown promise in reproducing PD-like pathology. However, current implementations of this SynFib model have largely been confined to rats and require sequential surgeries, which increase animal distress and reduce reproducibility. Here, we present a streamlined protocol to generate a SynFib mouse model of PD that integrates rAAV-α-synuclein delivery and PFF injection into a single stereotaxic surgery. Using fine glass capillaries, this method prevents backflow of injected material, reduces injection-induced trauma, minimizes neuroinflammation, and ensures robust lesion development. This streamlined mouse model provides a reproducible and practical system to investigate α-synuclein-associated pathology and serves as a versatile platform for preclinical testing of potential therapeutics for PD. Key features • This protocol optimizes the SynFib model of PD in mice and combines rAAV-driven α-synuclein expression and PFF injection in one stereotaxic surgery, reducing animal distress. • Uses modest α-synuclein expression levels targeted to cells in the substantia nigra region of the brain. • The mouse model recapitulates hallmark PD-like features, including α-synuclein aggregation, progressive neurodegeneration, and neuroinflammation. • This mouse model can be a useful tool for α-synuclein-related mechanistic studies and preclinical therapeutic testing for PD.

Keywords:  Dopaminergic neurons; Neurodegeneration; Parkinson’s disease; Preclinical model; Stereotaxic surgery; SynFib model; α-synuclein mouse model

DOI:  https://doi.org/10.21769/BioProtoc.5525