Cellular and animal models to investigate pathogenesis of amyloid aggregation in neurodegenerative diseases

Abnormal aggregation of amyloid proteins, e.g. amyloid β (Aβ), Tau and α-synuclein (α-syn), is closely associated with a variety of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Cellular and animal models are useful to explore the neuropathology of amyloid aggregates in disease initiation and progression. In this protocol, we describe detailed procedures for how to establish neuronal and PD mouse models to evaluate amyloid pathologies including self-propagation, cell-to-cell transmission, neurotoxicity, and impact on mouse motor and cognitive functions. We use α-syn, a key pathogenic protein in PD, as an example to demonstrate the application of the protocol, while it can be used to investigate the pathologies of other amyloid proteins as well. The established disease models are also useful to assess the activities of drug candidates for therapeutics of neurodegenerative diseases.


INTRODUCTION
Amyloid protein aggregation is a common pathological hallmark of neurodegenerative diseases, which plays essential roles in the initiation and progression of the diseases (Arai et al. 2006;Goedert et al. 1988;Murphy and LeVine 2010;Spillantini et al. 1997). For example, α-synuclein (α-syn) forms amyloid fibrils depositing into intraneuronal Lewy bodies/Lewy neurites (LBs/LNs) that serve as a histological hallmark of Parkinson's disease (PD) (Baba et al. 1998;Spillantini et al. 1997). Moreover, during the progression of PD, αsyn fibrils self-propagate and spread via cell-to-cell transmission across PD patients' brains (Luk et al. 2012), which cause degeneration of midbrain dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) (Damier et al. 1999) leading to bradykinesia, tremor, and postural instability (Luk et al. 2012). Studies have shown that α-syn preformed fibrils (PFFs) but not monomers induce the formation of LBs/LNs-like inclusions by recruiting and converting endogenous soluble monomeric α-syn protein into insoluble aggregates in primary neurons (Luk et al. 2009;Volpicelli-Daley et al. 2014). Moreover, administration of synthetic α-syn PFFs into mouse brain through stereotaxic injection leads to LBs/LNs formation in multiple brain regions and impairment of motor function (Zhang et al. 2019). Based on recent advances in cellular and animal model studies of amyloid fibrils (Long et al. 2021;Volpicelli-Daley et al. 2014;Zhang et al. 2019), we describe a series of assays to study α-syn fibril pathologies in cells and in vivo, which may help to understand the formation and the neurological effects of α-syn fibrils and further develop therapeutic drugs to cease PD progression.

Development of the protocol
The protocol is mainly composed of four parts, including α-syn PFFs preparation, cell viability assay, primary neuron model, and PD mouse model. The protocol provides a systematic pipeline to study the pathological properties of different α-syn fibril strains. For the preparation of α-syn PFFs, the concentration of fibrils needs to be directly and accurately measured since different amyloid proteins exhibit distinct fibril converting rates. Next, the propagation of α-syn PFFs in primary neurons is investigated by monitoring the neuronal aggregation of α-syn at different time points. α-Syn pathology is further evaluated in a PD-related mouse model induced by injecting exogenous α-syn PFFs. In sum, we provide a detailed protocol that is useful to study the pathology of α-syn amyloid fibrils in cells and in vivo.

Applications and advantages of the protocol
This is a systematic protocol for evaluating the pathological amyloid fibrils both in cells and in vivo. This protocol is not limited to α-syn study, but also can be applied to investigate neuropathological activities of other amyloid proteins such as Tau, TDP-43 (TAR DNAbinding protein 43kDa) and FUS (fused in sarcoma). This protocol describes the preparation and characterization of amyloid fibrils, measurement of the cytotoxicity of fibrils, endogenous aggregation and propagation induced by fibrils in primary neurons, and assessment of the fibril pathology in disease-related mouse models. These models are well-established with defined disease-related phenotypes and relatively easy for setup in biological laboratories following the detailed instructions described below. Moreover, these models provide important disease-related cellular and animal models to evaluate antibodies and inhibitors screened from in-vitro assay for the drug discovery of neurodegenerative diseases.

Limitations of the protocol
1 As for the mouse model, the stereotaxic injection site of fibrils in the brain, dorsal striatum (dSTR), described in this protocol is the most suitable for αsyn fibrils. For other pathological amyloid fibrils such as Tau fibrils and TDP-43 fibrils, different injection sites may need to be explored to better mimic their pathologies in diseased brains. 2 As for the behavioral tests, mice are easily affected by the environment and the tester. Consistency should be carefully controlled in experiments. 3 During the process of transcranial perfusion and frozen sectioning, man-made interferences are inevitable and should be considered. 4 Both the integrity and symmetry of brain slices influence the quality of immunofluorescence imaging. 5 Additional behavioral test assays may need to be performed including water maze, shuttle box experiment and balance beam experiment to evaluate the cognitive and motor dysfunction of the diseased mice.

Overview of the protocol
Firstly, the cytotoxicity of α-syn PFFs is examined by using SH-SY5Y neuroblastoma cell line with CCK-8 kit.
Next, exogenous α-syn PFFs inducing endogenous α-syn aggregates are studied in primary neurons, which is characterized by immunofluorescence staining. Then, mice are inoculated with α-syn PFFs leading to motor deficit and endogenous α-syn amyloid aggregates in vivo. Together, the protocol is applied to test the neurotoxicity and aggregation of α-syn fibrils both in cells and in vivo.

Cellular assay
1 Culture and plate SH-SY5Y cells for α-syn PFFs treatment. Cell viability is assessed by using the CCK-8 kit. 2 Preparation for primary neuron culturing, such as plate coating, medium setup, papain activation, and so on. 3 Dissect the cerebral cortex of E16-E18 rat embryos, and settle them in HBSS buffer on ice. 4 Digest the brain tissue with activated papain and DNase for 30 min. And then filter the brain tissue after digestion through a 40-mm nylon mesh cell strainer. 5 Resuspend the cell pellet and perform cell counting.
Plate cells as 10-15 × 10 4 cells/well and put plates into a culture incubator. 6 At the 8 th day in vitro (DIV), treat neurons with α-syn PFFs, incubate these neurons for desired days, and collect samples. 7 To stain primary neurons, we fix them with 4% paraformaldehyde (PFA) after rinsing with phosphate buffered saline (PBS). Then permeabilize neurons and block them. 8 Incubate coverslips with the primary antibody at 4 °C overnight followed by washing with PBST (0.1% Triton X-100 in PBS). 9 Secondary antibodies are incubated with neurons at room temperature (RT) for 1 h in a dark room followed by washing with PBST. 10 Mount coverslips on glass slides with the medium.

Disease-related mouse model
1 For stereotaxic injection, first weigh mice that are at an age of eight weeks. 2 Fix mice gently on the stereotaxic apparatus after anesthesia. 3 Shave the hair on the top of the mouse's head and then cut the scalp with scissors to expose the skull. 4 Position the brain area to be injected (dSTR for αsyn) and drill a hole at the target region. 5 Inject α-syn PFFs with a micro syringe. The required amount of PFFs for injection is calculated according to the weight of the mouse, 0.2 μg/g is suggested. 6 At 3-month post injection (mpi), behavioral tests are performed. Before the open field test (OFT), mice are handled for 3 d. 7 After OFT, mice need to rest for one day. 8 Then rotarod training and tests are performed. 9 After the mice rest for one day, pole tests are performed. 10 Then the mice are sacrificed for immunofluorescence staining experiments. Mice brains go through dehydration for three times with sucrose dissolved in PBS after perfusion and fixation. 11 Each brain tissue is serially sectioned into 30-μm slices with a cryostat microtome. 12 Brain slices are blocked with 10% goat serum and stained with primary antibodies and fluorescent secondary antibodies. 13 Mounting slices on glass slides with the medium. 14 Spinning disk fluorescence confocal microscope is required for imaging.

EXPERIMENTAL DESIGN α-Syn PFFs preparation
After the purification of the monomeric α-syn protein, filter the protein with a 0.22-μm centrifugal filter. Then α-syn fibril is prepared as previously reported (Li et al. 2018). Calculate the yield of α-syn fibril by subtracting the amount of residual soluble α-syn after pelleting the fibrils with the total amount of α-syn monomers. Then α-syn PFFs are produced by sonication with a probe tip sonicator. The morphologies of α-syn fibrils and PFFs are characterized by TEM. The PFF stock solution is stored at -80 °C.

Cell viability assay
Cell viability is measured by using a CCK-8 kit. SH-SY5Y cells are cultured and plated in a 96-well plate (6,000 cells/well, 100 μL/well). Then α-syn PFFs are applied to treat SH-SY5Y cells with final concentrations of 0, 0.01, 0.1 and 1 μmol/L (equivalent to monomer concentration) for 24 h. Further test the cell viability with CCK-8 kit following the manufacturer's protocol. Briefly, add 10 μL CCK-8 solution to each well, incubate the plate for 1-4 h in darkness. Finally, the absorbance of each well is measured at 450 nm.

Behavioral tests
At 1

Immunofluorescence staining
Mice are anesthetized with isoflurane vapor and transcranially perfused. The brains are fixed by 4% PFA and dehydrated with sucrose solution. Then the brains are sectioned into 30-μm coronal slices with cryostat microtome. Right before staining, brain slices are first blocked and then incubated with primary antibody at 4 °C overnight. The slices are further incubated in blocking buffer containing secondary antibody for 2 h at RT after being washed three times with PBS. Lastly, slices are mounted on glass slides with the mounting medium.

PROCEDURE α-Syn PFFs preparation [TIMING 8 d]
1 α-Syn protein is purified as previously described (Li et al. 2018) and is filtered with the 0.22-μm centrifugal filter to remove precipitates. 2 To incubate fibril, α-syn protein (200 μmol/L, 200 μL in 50 mmol/L Tris, pH 7.5, 150 mmol/L KCl) in a 1.5-mL microtube is placed in the ThermoMixer, at 37 °C with constant agitation (900 r/min) for 7 d. 3 After the maturation of α-syn fibril, the morphology of fibrils is characterized by TEM. 4 α-Syn fibril is centrifuged at 14,500 r/min, 25 °C for 40-50 min. Then remove the supernatant (volume of supernatant, V s ) and measure the concentration of residual soluble α-syn (C s ). The amount of α-syn fibril is calibrated by subtracting the amount of residual soluble α-syn (C s × V s ) with the total amount of α-syn monomer (C t × V t ). The equation for the concentration of α-syn fibril (C p ) is . 5 α-Syn fibril is resuspended with sterile PBS to a final concentration of 100 μmol/L, and sonicated into αsyn PFFs with a probe tip sonicator, with 20% power, 1 s on, 1 s off, total time 30 s. The morphology of PFFs is characterized by TEM.
[TIP] Divide PFFs into aliquots and store them at -80 °C to avoid multiple freeze-thaw cycles.

Cell viability test [TIMING 2-5 d]
6 SH-SY5Y cells culture. Maintain cells with DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) in the 37 °C, 5% CO 2 cell culture incubator. 7 Plating. Trypsin-digested SH-SY5Y cells (6, [TIP] If ear rods are fixed on the respiratory center, the lower limbs of mice would jump up. During surgery, the anesthetized mouse is placed on a heating pad to maintain body temperature at 37 °C. 22 Shaving. Use a razor to shave the hair on the top of the mouse's head, apply depilatory cream for a short while, and then wipe the hair with a cotton swab. [TIP] While waiting for the depilatory cream to work, apply erythromycin ointment on the eyes of mice. Put ear studs on the mice. Change the cranial drill bit. 23 Anatomy. Lift the scalp with tweezers and cut it vertically with scissors (from the eyes to the base of the ears, around 1 cm long). Corrode the meninges with hydrogen peroxide (H 2 O 2 ) for 10 s, and then wipe clean with a cotton swab dipped in saline.
[TIP] The cranium is fully exposed, and Bregma and Lambda are clearly shown under the effect of H 2 O 2 . Find Bregma and Lambda on the surface of the skull in the eyepiece.
[?TROUBLESHOOTING] 24 Location. Location procedure follows the protocol published previously (Long et al. 2021). Details are as following. (A) X-axis leveling i. First, make a rough adjustment with the naked eye; ii. Move and position the syringe needle to touch the Bregma on the surface of the skull, and reset the x, y, and z values of the desktop digital display to zero. iii. Move the syringe needle to the right to the point where x = 2.0, and record z 1 value; move the electrode to the left to the point where x = -2.0, and record z 2 value. Leveling the left and right of the mouse brain to make |z 1 -z 2 | ≤ 0.03. iv. After every adjustment of the position of the mouse brain, repeat Steps ii and iii.
[TIP] Before leveling, poke the skull with tweezers to make sure it will not move.
(B) Y-axis leveling i. Move the syringe needle from Bregma (0, 0, 0) to Lambda, and record the y L (Lambda of mature mice should be (0, -4.2, z L )) theoretically. If the mouse is retarded, then y < 4.2, the injection position should be adjusted in the same proportion, that is (x, y, z) × y L /4.2. ii. When the electrode is moved from Bregma to Lambda, recording the z L (z value of Lambda). Leveling the Y-axis direction of the mouse brain to make |0-z L | ≤ 0.03. iii. After every adjustment of the position of the mouse brain, repeat Steps (A) and (B). 25 Injection (A) Locate the target brain area according to the x, y, z position of the target brain area, for example, dSTR (2.0, ±0.2, -2.6), and mark it with a marker. (B) Drill a hole at the marking site with a cranial drill, drill once and align the electrode to see if the position is correct, repeat three times. Then drill for 2 s and stop for 1 s until the drill succeeds.
[TIP] Drill the hole vertically as much as possible; a little cerebrospinal fluid will leak out after drilling and wipe it clean, avoid bleeding; if both hemispheres are injected, drill both sides.
[?TROUBLESHOOTING] (C) Calculate the dosage of α-syn PFFs (2 μg/μL) according to the weight of each mouse (0.2 μg/g), and suck the sample into the syringe with a micro syringe pump (parameters: set up, total volume: n μL, speed rate: 1 μL/min, mode: withdraw). (D) On the surface of the hole drilled, set z to 0 and drop the electrode to the target position. Set the parameters of the micro syringe pump. Volume: 0.2 μL, rate: 0.5 μL/min, mode: infuse and start; then adjust to 0.2 μL/min, n -0.2 μL.
[CRITICAL STEP] After injection, keep the micro syringe still for 5-6 min to prevent α-syn PFFs/PBS overflowing.
Stitch the ends firstly, and then sew the middle. Finally, apply antibiotics to the wound and wipe off the eye ointment.
[?TROUBLESHOOTING] 27 Transfer the mice into a recovery cage under a warming lamp until mice wake up.
Behavioral tests  After injection, leave the mice under the same living conditions for three months.

Open field test [TIMING 2-3 h for 24 mice]
29 Adjust and test the instruments. For example, fix the camera on the ceiling and setting the light inside each chamber at 30-35 lux. 30 Put each mouse in the middle of the chamber (40 cm × 40 cm) for 10 min. Their activities are monitored and recorded by the camera and analyzed by EthoVision XT (Noldus11.5).
[TIP] Researchers need to leave the room to reduce human interferences. Change the chamber of the same group and clean the chamber with 75% alcohol between each trial.

Rotarod training [TIMING 3 h/d for 24 mice, 3-5 d]
31 Train the mice twice for 2 min with constant speed, 4 r/min. The 3 rd trial goes on with accelerating speed from 0 to 40 r/min in 1.5 min, total of 2 min. Record the total duration of the mouse on the rotating rod. There is a 3-min interval between each trial. All mice are trained for 3-5 d consecutive days until most mice perform well. REST [TIMING 1 d] 32 Mice receive two tests continuously on the rotarod expediting from 0 to 40 r/min in 1.5 min, then keep at 40 r/min for another 3.5 min. Record the total duration of the mouse on the rotating rod.
[TIP] Clean the rotarod with 75% alcohol between each trial. At least three consecutive or discontinuous turns of the mouse following the rotarod are deemed as landing.

Pole test [TIMING 3 h for 24 mice]
33 Climbing. Place each mouse on the top of the metal rod (length: 50 cm; OD: 1 cm) that is wrapped with medical tape. Guide the mouse head facing downwards. Train all mice to climb down the rod once before testing for five times continuously and record the climb-down duration time. After dehydration, put all brains into embedding boxes filled with optimal cutting temperature compound (OCT) and rapidly freeze at -80 °C.
[TIP] The ratio of antibodies used can be optimized. 41 Washing. Brain slices are then rinsed in PBST, 10 min/time for three times and once more with PBS. 42 Secondary antibody incubation. All brain slices are incubated with the secondary antibodies diluted with the blocking solution containing goat anti-rabbit Alexa Fluor 568 (1:1000, abcam, ab175471) and goat anti-rat Alexa Fluor 488 (1:1000, abcam, ab150157) for 2 h at RT.
[TIP] The ratio of antibodies used can be optimized. 43 Washing. Brain slices are then rinsed in PBST, 10 min/time for three times and once more with PBS. 44 The slices are mounted on glass slides with Prolong gold antifade reagent to preserve fluorescence signal. Store at 4 °C.
Confocal imaging [TIMING 10 d] 45 Imaging. A spinning disk fluorescence confocal microscope is required to capture fluorescence images. Two kinds of selective lens patterns are applied, 20× (NA = 0.75) air objective for SN and 40× (NA = 1.25) water immersion objective for STR. Lasers of 630, 561 and 488 nm are used to excite Alexa Flour 647, 568 and 488, respectively and sequentially. 46 Image analysis and statistical analysis.

[?TROUBLESHOOTING]
Troubleshooting advices can be found in Table 1.

ANTICIPATED RESULTS
1 Following the protocol, we first prepared α-syn PFFs which were characterized by TEM. Figure 1A shows the operating process. The homogeneous and unbranched fibrils were imaged by TEM (Fig. 1B). α-Syn fibrils were sonicated into α-syn PFFs, which were well dispersed and uniform in size (Fig. 1C). 2 The SH-SY5Y cells cultured in a 96-well plate were treated with sterilized α-syn PFFs with final concentrations of 0, 0.01, 0.1, 1 μmol/L for 24 h ( Fig. 2A). The result showed that the cell viability decreased with increasing concentrations of PFFs (Fig. 2B). Next, we cultured rat primary neurons and treated them with sterilized α-syn PFFs at 8 DIV with a final concentration of 100 nmol/L (Fig. 3A). Then the neurons were further incubated until 22 DIV, 26 DIV, 30 DIV and so on. Endogenous α-syn aggregates were induced by the addition of α-syn PFFs with increasing amounts over time in primary neurons (Fig. 3B). These results demonstrate the transmission of α-syn PFFs from medium to cell interior and the propagation of α-syn PFFs in cells.

To investigate the neuropathology induced by α-syn
PFFs in vivo, PFFs were bilaterally inoculated into the  (Fig. 4A). The data of OFT showed that there was no difference between α-syn PFFs-injected and PBS-injected mice (Fig. 4C). However, mice inoculated with α-syn PFFs performed worse than PBS-injected control mice in the rotarod and pole test (Fig. 4D, E). We further cryo-sectioned the brain tissues of the mice into 30-μm coronal slices and examined α-syn pathology at this time point by immunofluorescence staining (Fig. 5A). Images of dSTR showed obvious α-syn aggregates in the group of PFFs-injected mice (Fig. 5B). Furthermore, the aggregates were also observed in the substantia nigra (SN) (Fig. 5B).     5 Immunofluorescence staining of PD-related mouse brain. A Workflow of immunofluorescence staining of PD-related mouse brain. B Representative data of the immunostaining. Images of dSTR (top) and SN (bottom) 3-month post inject are shown respectively. Antibodies were used to mark p-α-syn (red) and DAT (green) Cellular and animal models to investigate amyloid aggregation PROTOCOL These results demonstrate that α-syn PFFs efficiently induced endogenous aggregates and α-syn pathology in vivo.

Reagents
• Source materials, e.g., tissues and organs for primary neuron culture, are from Sprague-Dawley rats. Mice for establishing PD models are C57 BL/6J mice.
[CAUTION!] All experiments using animals must be carried out according to relevant governmental and institutional regulatory guidelines.
dissolved. Bring the volume to 500 mL and stir until fully dissolved. This solution can be stored at 4 °C for three months.
• Sucrose (30%). Dissolve 150 g sucrose in 400 mL deionized water and stir until most of it is dissolved. Bring the volume to 500 mL and stir until fully dissolved. This solution can be stored at 4 °C for three months.
• Borate buffer (0.1 mol/L). Dissolve 1.24 g boric acid and 1.90 g borax in 400 mL deionized water and stir until most of it is dissolved. Adjust the pH to 8.5 with NaOH. Bring the volume to 500 mL and filtersterilize the solution. This solution can be stored at 4 °C. prepared figures; and HL, SZ and DL wrote the manuscript. All authors read and approved the final manuscript.

Compliance with Ethical Standards
Conflict of interest Houfang Long, Shuyi Zeng and Dan Li declare that they have no conflict of interest.
Human and animal rights and informed consent All institutional and national guidelines for the care and use of laboratory animals were followed.
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