Modeling neurodegeneration using pre-formed fibrils (PFFs)

Protein aggregation is a key characteristic of neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD). In these pathological states, proteins like Tau, Amyloid beta, alpha-synuclein, TDP-43, and Huntingtin form various unwanted structures, from oligomers and prefibrillar assemblies to highly ordered aggregates.

The fibrillar stage of these proteins signifies a rapid growth phase in which fibrils actively recruit monomers for elongation. Furthermore, these fibrils can fragment randomly, creating "seeds" that spread to other cells, independently attracting monomers to develop new fibrils.

Pre-formed fibrils (PFFs) are active fibrils created in vitro, possessing "seeding" capabilities. They continuously attract and recruit soluble endogenous pathological proteins, leading to the formation of aggregates and ultimately triggering neurodegenerative pathologies.

Schematic presentation of protein misfolding and aggregation. Image Credit: ACROBiosystems

Establishing reliable disease models is essential for uncovering pathological mechanisms, evaluating therapeutic interventions, and assessing the safety of drug candidates.

Compared to traditional disease modeling approaches, PFFs-induced pathology does not rely on gene editing, chemical alterations, or physical damage, making it more effective at mimicking naturally occurring pathological processes. Consequently, PFFs represent a novel and promising approach to modeling neurodegenerative diseases.

Pre-formed fibrils (PFFs) production and validation

PFFs can be generated from monomers either by incubating at 37 °C with shaking or by induction with heparin. Successful experiments depend on meticulous quality control and precise initial preparation.

A critical factor in the successful induction of PFFs is using high-quality monomers that are highly pure, concentrated, and correctly conformed. Additionally, controlling endotoxin levels is vital since PFFs are used in cellular and animal experiments. Before use, PFFs should be sonicated to a length of 50 nm or shorter to ensure their recruiting activity and facilitate endocytosis.

The morphology and activity of PFFs can be verified using an electron microscope and a thioflavin T (ThT) fluorescence assay, respectively. Successfully induced PFFs display a fibril structure under an electron microscope.

The ThT assay, a classic method for detecting β-sheet structures, measures PFF activity. As PFFs recruit more monomers, they acquire more β-sheet structures, and when ThT binds to these structures, the fluorescence value increases, reflecting the activity of the PFFs.

Pre-formed fibrils (PFFs) product

As the neuroscience-focused brand of ACROBiosystems, Aneuro offers a range of PFFs, including Tau-441, alpha-synuclein, Amyloid beta, TDP-43, and SOD-1 PFFs. These products support and accelerate the development of reliable neurodegenerative disease models.

PFFs product list

Source: ACROBiosystems

More PFFs relevant products under development. PFFs are under development.

PFFs product features

• Produced by high-quality monomers, the purity and homogeneity of the monomer are≥90%, as verified by MALS, which is more conducive to the formation of PFFs and ensures their activity.
• The aggregation morphology and recruitment activity of PFFs are verified by electron microscopy and ThT fluorescence assay. Free protocols are shared.
• Lower endotoxin: Monomer endotoxin ≤ 1.0 EU/μg, suitable for various in vitro and in vivo experiments.
• Stable lead time, strict control to ensure high batch-to-batch consistency, highly cost-effective.
• Customized products and services are provided with a variety of fluorescent labeling solutions: star staining, biotin labeling, chemical labeling, etc.

PFFs product verification data

PFFs morphology (electron microscopy)

Under electron microscopy, the PFFs exhibit unique fibrous structures, as illustrated in the image below, proving the accuracy of the PFF products’ morphology.

TEM of Human Tau-441/2N4R Pre-formed Fibrils Protein (Cat. No. TAU-H5115). Image Credit: ACROBiosystems

TEM of Human Tau-441 K18 Pre-formed Fibrils Protein (Cat. No. TAU-H5146). Image Credit: ACROBiosystems

TEM of Human Tau-441 K18 (P301L) Pre-formed Fibrils Protein (Cat. No. TAU-H5113). Image Credit: ACROBiosystems

PFF bioactivity (cell base) is consistent with the reference

Tau PFFs (TAU-H5115) caused considerable protein aggregation in the cytoplasm of the HEK293/Human Tau-K18 (GFP) Stable Cell Line (Cat. No. CHEK-ATP087).

HEK293/Human Tau(GFP) Stable Cell Line were transduced with Human Tau-441 / 2N4R Pre-formed Fibrils Protein, Tag Free (ThT active) (Cat. No. TAU-H5115) and Human Tau-441 / 2N4R Protein, Tag Free (MALS verified) (Cat. No. TAU-H5117) respectively. The fluorescence of GFP-Tau (Green) and DAPI (Blue) were detected by confocal microscope. A. Lipo2000 transduction. B. Lipo2000 and Human Tau-441 / 2N4R Protein, Tag Free (MALS verified) transduction. C. Lipo2000 and Human Tau-441 / 2N4R Pre-formed Fibrils Protein, Tag Free (ThT active) transduction. Scale bars, 50 μm. Image Credit: ACROBiosystems

Tau PFFs (TAU-H5146) caused considerable protein aggregation in the cytoplasm of the HEK293/Human Tau (GFP) Stable Cell Line (Cat. No. CHEK-ATP087).

HEK293/Human Tau (GFP) Stable Cell Line (Cat. No. CHEK-ATP087) were transduced with Human Tau-441 K18 Pre-formed Fibrils Protein, His Tag (Cat. No. TAU-H5146) and Human Tau-441 K18 Protein, His Tag respectively. The fluorescence of GFP-Tau (Green) was detected bA. Lipo2000 transduction. B. Lipo2000 and Human Tau-441 K18 Protein, His Tag transduction. C. Lipo2000 and Human Tau-441 K18 Pre-formed Fibrils Protein, His Tag transduction (Routinely tested). Image Credit: ACROBiosystems

PFFs activity (ThT assay)

PFFs caused quicker aggregation of monomer proteins than the control group that only added monomer.

Thioflavin T emission curves show increased fluorescence (correlated to tau aggregation) over time when tau wild-type monomers (Cat. No. TAU-H5117) are combined with tau wild-type Pre-formed Fibrils (Cat. No. TAU-H5115). Image Credit: ACROBiosystems

Thioflavin T emission curves show increased fluorescence (correlated to tau aggregation) over time when tau K18 monomers are combined with Human Tau-441 K18 Pre-formed Fibrils Protein, His Tag (Cat. No. TAU-H5146) (QC tested). Image Credit: ACROBiosystems