Stefan G. D. Rudiger

A new molecular mechanism explains how phosphorylation and acidosis act together to drive Tau aggregation. Ser352 phosphorylation forms Tau condensates at pH 7.1, while a pH drop triggers their irreversible transition into amyloid filaments.

Huntington’s disease (HD) is caused by expansion of a polyglutamine tract in the huntingtin (Htt) protein, leading to aggregation of the exon 1 fragment (HttEx1) into amyloid fibrils. HttEx1 forms one of the lowest-complexity amyloid cores known, its fibril core consists of a single amino acid, glutamine. With emerging therapies improving patients’ prospects by silencing expression of HTT, tools to monitor HttEx1 aggregation become essential for timely intervention and next-generation therapeutics. Here, we show that the peptide FibrilPaint1 selectively binds HttEx1Q44 fibrils without interacting with monomeric protein, allowing to measure and trace HttEx1 amyloid fibrils. Using the FibrilRuler assay, we tracked fibril formation from early species to larger clustered assemblies. The non-fluorescent variant, FibrilPaint20, was used to recruit the E3 ubiquitin ligase CHIP to HttEx1 fibrils, enabling site-specific ubiquitin tagging. However, unlike Tau fibrils, ubiquitinated HttEx1 fibrils resisted proteasomal degradation. This reveals a fundamental difference in how amyloids with extremely low–complexity cores respond to cellular clearance machinery. Together, our findings establish the FibrilPaint peptide family as a toolset for the detection and molecular targeting of amyloids, providing new opportunities to study protein aggregation and act as building blocks for future diagnostic and therapeutic strategies in neurodegenerative diseases. Highlights: FibrilPaint1 selectively binds HttEx1Q44 amyloid fibrils and allows monitoring of fibril growth using the hydrodynamic radius (FibrilRuler). FibrilPaint20 recruits the E3 ligase CHIP to Htt fibrils, enabling ubiquitination. Despite successful ubiquitination, Htt fibrils resist proteasomal degradation in vitro, highlighting structural barriers. FibrilPaint provides a scaffold for functional targeting of amyloids with diagnostic and therapeutic potential. Graphical Graphical abstract The FibrilRuler Test: FibrilPaint enables measurement of Huntingtin fibril size during aggregation: After a short lag-phase following removal of the protective MBP tag by Factor Xa, fibrillation proceeds rapidly. Subsequent fibril clustering further accelerates growth, leading to exponential increases in aggregate size.

Tau aggregation into amyloid fibrils is linked to the development of neurodegenerative diseases, including Alzheimer’s disease (AD). The molecular processes driving aggregation in disease are still being uncovered, highlighting the need for innovative tools to study aggregation reactions. Here, we introduce FibrilPaint1 as a tool to measure the size of Tau amyloid fibrils in fluids, from early aggregation stages to mature fibrils. FibrilPaint1 is a 22mer peptide with exciting properties: i) FibrilPaint1 binds fibrils with nanomolar affinity; ii) it also binds to precursors, down to a size of only 4 layers; iii) it does not bind to monomers; iv) it is fluorescently labeled, which allows monitoring and localizing interactions; v) FibrilPaint1 recognizes various Tau fibrils, including patient-derived fibrils from AD, corticobasal degeneration (CBD), and frontotemporal dementia (FTD); vi) it also binds to fibrils from amyloids derived from Amyloid-β, α-synuclein, and huntingtin vii) FibrilPaint1 is selective for the amyloid state and does not have background binding to amorphous aggregates, blood serum, or cell lysate. In combination with flow-induced dispersion analysis (FIDA), a microfluidics technology, we determined the molecular size of amyloid fibrils with submicroliter sample volumes. This setup acts as a molecular ruler at layer resolution—we determined Tau fibril length from 4 to 1100 layers in solution. This is an interesting parameter for molecular studies in dementia, with potential for diagnostic applications.

Our study shows, by using synthetic multiphosphorylated peptide libraries that specific phosphorylation patterns govern the decision between aggregation and condensation of the R4 domain of the Tau protein.

Neurodegenerative diseases are characterised by the formation and accumulation of protein fibrils. The mechanism underlaying this aggregation process remains poorly understood. Fibril fragmentation, resulting in seed generation, plays a role in toxicity. Here we provide a quantitative picture of the impact of ultrasound on patient-derived and recombinant fibrils from various diseases. Fragmentation of recombinant Tau fibrils and patient-derived fibrils from Alzheimer’s Disease, Corticobasal Degeneration and Frontotemporal Dementia generates amyloid and non-amyloid species. Interestingly, patient-derived fibrils are more susceptible to ultrasound than artificial fibrils. Understanding fibril fragmentation and the generation and nature of seeds may provide insights to the molecular mechanism of the disease progression, contributing to the development therapeutic approaches.

Summary: Failure of the Protein Quality Control (PQC) System to control the Tau protein leads to amyloid fibrils that are at the basis of various Tauopathies, including Alzheimer’s Disease (AD). Reinstating the PQC for Tau is desirable. Here we show that the PROTAC peptide FibrilPaint20 (FP20) recruits the ubiquitination system to Tau amyloid fibrils. We reconstituted the ubiquitin-proteasome cascade and monitored the stepwise ubiquitin transfer using the microfluidics technique FIDA. To direct ubiquitination specifically to amyloids, we discovered FP20, a bifunctional peptide that connects amyloid fibrils to the E3 ligase CHIP. FP20 mediated ubiquitination of recombinant and patient derived fibrils from various tauopathies, ultimately enabling the 26S proteasome to target CHIP-ubiquitinated Tau fibrils. This shows that FP20 cooperates with the PQC system and can initiate fibril cleavage. This offers a strategy to target amyloid diseases by cooperation with the PQC system exploiting key cellular systems.

Amyloid fibrils are a common pathological hallmark in multiple neurodegenerative diseases, yet molecular tools to selectively recognise and manipulate them remain scarce. We report FibrilPaints, a family of modular peptides designed for selective amyloid binding and adaptable chemical functionality. The degenerative amyloid-targeting unit of FibrilPaints, W ₅ P ₄ H ₃ R ₂ , has a high content of π-stacking and aromatic side chains. Systematic sequence variation, altering charge, termini, and residue order, revealed the importance of the composition of the amyloid-targeting unit for high-affinity binding across Tau and Huntingtin fibrils. Importantly, sequence changes outside this unit do not preclude fibril binding, which permits attachment of fluorophores or E3-recruiting motifs for targeted protein degradation. This work establishes FibrilPaint as a modular peptide system for the detection and modulation of amyloids. Abstract Figure: Figure of content: The modular design of FibrilPaints enables systematic evaluation of their functionality by testing the binding capacity of each variant (FibrilPaintX) to distinct amyloid fibrils. Successful binding results in visible ‘painting’ of the fibrils, facilitating their detection and downstream research.

Background: Protein aggregation is highly related to the development of Alzheimer’s Disease. The formation of extracellular beta‐amyloid plaques and intracellular neurofibrillary tangles, composed by Tau fibrils, leads to neuronal dysfunction and degeneration. Current diagnosis techniques, such as brain imaging and cerebrospinal fluid tests, can only determine the formation of the protein deposits, which happens to be too late. It is of great importance to visualize initial aggregation steps, which is a prerequisite for early diagnosis.Method: Tau fibrils were extracted from AD patient‐derived brains or generated in vitro, and analysed by Flow Induced Dispersion Analysis with WH peptide.Results: Here we present a method to monitor Tau fibrils size from the first aggregation steps. We designed WH peptide as a biocompatible, fluorescence‐labelled compound that specifically recognizes protein fibrils but not non‐amyloidic monomers, based on specific chemical properties, so called π‐stacking and H‐bonding interactions. These interactions are crucial for the recognition of aggregated species. In combination with Flow Induced Dispersion Analysis, a novel microfluidics technology, we determined the size of the aggregated species with sub‐microliter sample volumes. It is a key challenge in the field to study patient derived fibril. WH peptide labels Alzheimer’s Disease patient‐derived fibrils, which structurally differ from the in vitro generated ones, which allowed us to determine the size of the ex vivo fibrils.Conclusion: WH peptide fluorescently labels Alzheimer’s Disease patient‐derived fibrils. WH peptide is biocompatible and could be further functionalized for imaging applications, which could be a basis for early‐stage diagnosis of Alzheimer’s Disease.

Tau aggregation into amyloid fibrils is linked to the development of neurodegenerative diseases, including Alzheimer’s Disease. The molecular processes underlying aggregation in disease are poorly understood. Here, we introduce FibrilPaint1 as a tool to measure the size of Tau amyloid fibrils in fluids, from early aggregation stages to mature fibrils. FibrilPaint1 is a 22mer peptide with many exciting properties, which makes it a tool for diagnostics and an attractive start point for developing a class of effective fibril targeting degraders: (i) FibrilPaint1 binds fibrils with nanomolar affinity; (ii) it does also bind to oligomeric precursors, down to a size of only 4 layers; (iii) it does not bind to monomers (KD > 100 µM); (iv) it is fluorescently labelled, which allows monitoring and localising interactions. (v) FibrilPaint1 recognises various Tau fibrils, including patient derived fibrils from Alzheimer, Corticobasal degeneration and Frontotemporal dementia; (vi) FibrilPaint1 is selective for the amyloid state and does not have background binding to amorphous aggregates, blood serum or cell lysate. In combination with Flow Induced Dispersion Analysis (FIDA), a microfluidics technology, we determined the molecular size of amyloid fibrils with sub-microliter sample volumes. This set-up acts as a molecular ruler at layer resolution - we determined Tau fibril length from 4 to 1100 layers in solution. This is an interesting parameter that can be used for diagnostic applications and biochemical research in dementia.