Understanding tau PFFs and alpha-synuclein PFFs

The misfolded proteins tau and α-syn cause irregular aggregation and spread in neurodegenerative illnesses, including Alzheimer’s disease (AD) and Parkinson’s disease (PD).

In AD, tau protein becomes hyperphosphorylated, impairing its microtubule-binding activity, resulting in microtubule destabilization, neurofibrillary tangle development, and neuronal death.¹

In PD, α-syn aggregates into Lewy bodies within nigrostriatal dopaminergic neurons, interrupting normal function and decreasing synaptic transmission.²

Tau and α-syn are linked to prion-like self-propagation, in which misfolded proteins act as templates, causing pathogenic proteins to misfold further and spread in the brain.³

As neurons degenerate in the central nervous system (CNS), the accumulation and transmission of misfolded proteins cause autonomic dysfunction and cognitive and motor impairments.

Pre-formed fibrils (PFFs) of tau and α-syn are extensively utilized in models to research AD and PD development. These fibrils act as seeds, causing further aggregation in vitro and in vivo, simulating illness progression, and enabling research into aggregation processes and possible therapeutics.^4,5

This article examines the morphology of tau and α-syn PFFs before using them in cellular models to investigate prion-like features in neurodegenerative disorders.

Discussion and results

Tau isoforms (0N3R, 0N4R, 1N3R, 1N4R, 2N3R, 2N4R) are categorized by amino-terminal exon inserts and carboxyl-terminal microtubule-binding domain (MBD) repeats.⁶ The MBD’s repetition regions contain hexapeptide motifs that enhance β-sheet formation, which is important for aggregation.

Tau isoform expression varies with developmental stage, with 0N present in fetal brains and various isoforms in adult brains.⁷ Full-length tau aggregation is usually slow, so studies often use the aggregation-prone MBD construct K18 or the P301L mutation to promote β-sheet formation.⁸

α-syn isoforms, particularly α-syn-112 and α-syn-98, aggregate quicker than the most prevalent isoform, α-syn-140.⁹ The A53T α-syn mutation causes neurotoxicity and is associated with early-onset PD.¹⁰

Morphology of tau and α-syn PFFs

This study utilized monomeric tau and α-syn for PFF synthesis. Following centrifugation, the supernatant was collected for protein concentration measurement, while a tiny fraction of the monomers were kept as negative controls.

The protein solutions were concentrated to 2–5 mg/mL and incubated at 37 °C with shaking for seven days to stimulate PFF production. The morphology of wildtype and mutant tau and α-syn PFFs were then analyzed using transmission electron microscopy (TEM).

Figure 1 illustrates the fibril shape of tau-2N4R (TAU-H5115), P301L mutant (TAU-H5113), α-syn-140 (ALN-H5115), and A53T mutant (ALN-H5114). Under negative stain TEM, fibrils ranged in diameter from 100 to 500 nm, with most appearing straight and with occasional twists.

Wild-type and mutant tau and α-syn fibrils formed thin and lengthy networks despite differing aggregation patterns.

Figure 1. TEM images of tau and α-syn PFFs in wild-type and mutant forms. (A) tau WT PFFs (TAU-H5115); (B) Tau K18 P301L mutant PFFs (TAU-H5113); (C) α-syn WT PFFs (ALN-H5115); (D) α-syn A53T mutant PFFs (ALN-H5114). Scale Bar, 200 nm. Image Credit: ACROBiosystems

Aggregation kinetics of tau and α-syn PFFs

The aggregation dynamics of tau and α-syn PFFs were studied using a Thioflavin T (ThT) fluorescence assay, which detects β-sheet-rich protein aggregates with high sensitivity.

In the tau ThT assay (Figure 2A), tau monomers (blue curve) aggregated slowly, with fluorescence increasing after 25 hours. Adding PFF (red curve) significantly expedited the aggregation process by shortening the lag period and increasing fluorescence intensity.

Tau PFFs (orange-red curve) exhibited steady fluorescence throughout the experiment, with no notable variations. In the α-syn ThT assay (Figure 2B), larger quantities of α-syn PFFs resulted in faster aggregation (blue and green curves), with shorter lag phases and increased fluorescence.

α-syn monomers (yellow curve) exhibited a slower rise in fluorescence, indicating delayed aggregation. The phosphate-buffered saline (PBS) control group showed no fluorescence increase in tau or α-syn ThT tests, indicating no aggregation.

The results show that tau and α-syn PFFs increase aggregation more than their respective monomers.

Figure 2. ThT seeding assay for tau and α-syn PFF. The emission curves show rising fluorescence over time, indicating aggregation when tau (TAU-H5117) and α-syn (ALN-H5214) monomers are added to tau PFFs (TAU-H5115) and α-syn PFFs (ALN-H5115), respectively. Image Credit: ACROBiosystems

In Vitro modeling of tau and α-syn pathology

To examine the pathophysiology of tau and α-syn, stable HEK293 cell lines expressing GFP-tagged 2N4R tau (amino acids 244-372, CHEK-ATP087) and full-length GFP-tagged α-syn (CHEK-ATP085) were used.

These cells rarely develop inclusions unless exposed to exogenous fibrils or undergo extended passaging. Using confocal microscopy, cells transfected with wild-type and mutant tau and α-syn PFFs formed nuclear speckles and juxtanuclear inclusion bodies.

Lipofectamine 2000 (Lipo2000)-mediated transduction increased the aggregation of tau and α-syn proteins, with mutant PFFs resulting in more inclusion bodies than wild-type (Figure 3A). This indicates that protein mutations promote aggregation and toxicity.

Cells with inclusion bodies form confined clusters, so the aggregated state is inherited stably. To test whether transfected cells behaved similarly, the cell lines with tau and α-syn inclusion bodies were identified and grown on 96- or 384-well plates.

The proportion of cells with inclusion bodies was quantified using high-content microscopy.

The findings indicate that the inclusion body-positive shape was maintained across multiple passages, indicating sustained propagation of tau and α-syn aggregates (Figure 3B). This approach successfully detected very hazardous cells expressing inclusion bodies.

Figure 3. PFFs induce aggregation in cellular models. (A) Quantitative analysis of protein aggregation scores across different groups treated with PFFs of α-syn (ALN-H5114, ALN-H5115) and tau (TAU-H5113) or GFP controls, with and without lipid-based transfection agents. (B) Representative fluorescence images showing the progression of α-syn and tau aggregation in cellular models after two rounds of treatment with PFFs. Image Credit: ACROBiosystems

Conclusion

This study shows that tau and α-syn PFFs successfully promote aggregation in HEK293 cell lines expressing GFP-tagged tau and α-synuclein. This provides a model for studying neurodegenerative illnesses, including AD and PD.

Both wild-type and mutant PFFs induce aggregation, with mutant versions (tau P301L and α-syn A53T) resulting in more severe pathology and higher toxicity in clinical cases.

Lipo2000 dramatically increased the effectiveness of PFF transduction by enhancing endogenous protein aggregation. Tau and α-syn aggregates pass down through cell generations, indicating their long-term presence and potential impact on disease progression.

These findings highlight the value of PFFs in researching protein aggregation mechanisms and screening medicinal drugs that target aggregation and its progression.

More research in more complicated models, such as organoids and animal systems, is needed to mimic human illness situations better and increase our understanding of neurodegenerative disease pathophysiology.

Methods and materials

Cell culture

To culture 293T cells, warm PBS, trypsin, and Dulbecco’s modified eagle medium (DMEM) media (10 % FBS, 1 % penicillin-streptomycin) before passing. After aspirating the culture medium, cells were washed with PBS before being digested with trypsin at 37 °C for five minutes.

DMEM was added to inhibit digestion, and the cells were centrifuged at 800 rpm for three minutes. The pellet was resuspended in DMEM and transferred to a fresh dish for further culture.

PFFs preparation

Tau and α-syn PFFs were created by diluting monomers to 2–5 mg/ml concentrations. The protein solutions were kept at 37 °C with continual shaking for seven days. The quality and shape of fibrils were evaluated with TEM.

ThT assays

ThT (25 μM) was produced in PBS and applied to protein samples in a 96-well plate (100 μL each). PFFs (9 μM) or monomers (120 μM) (or both) were incubated at 37 °C with 200 rpm. To track aggregation, measure fluorescence with a CLARIOstar plate reader every 1–72 hours at 450 nm excitation and 485 nm emission.

PFF transduction

The cells were transduced with PFFs using Lipofectamine 2000 (Invitrogen), and 100 μL Opti-MEM was incubated with 8 μL Lipo2000 and an equal volume of sonicated PFFs for 25 minutes at room temperature. The combination was applied to cells in a 12-well plate and incubated at 37 °C with 5 % CO₂ for 48 hours.

Aggregation detection

Following transduction, cells were rinsed with PBS and fixed in 4 % paraformaldehyde for 15 minutes. Confocal microscopy was used to detect the aggregated GFP-tagged tau and α-syn. The number and shape of inclusions were counted using high-content microscopy.

References

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