Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

Summary

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83^+/-:Gfap-luc^+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

Abstract

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83^+/-:Gfap-luc^+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83^+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83^+/-:Gfap-luc^+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83^+/-:Gfap-luc^+/-) mice with a median incubation time of 229 ±17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83^+/-:Gfap-luc^+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Introduction

There is growing evidence that alpha-synuclein has characteristics that are similar to those of the prion protein, particularly in its capacity to self-seed and propagate misfolding between cells and along neuronal pathways. This property of alpha-synuclein is also referred to as 'prion-like' or 'prionoid', and is supported by observations in transplantation experiments, which suggest the transmissibility of misfolded alpha-synuclein from diseased neurons to newly transplanted healthy neurons^1,2,3,4. Also direct injection of misfolded alpha-synuclein into the brain or the periphery, e.g. the hindlimb muscle or intestinal wall, results in a spread of alpha-synuclein pathology to distal parts of the CNS^5,6,7,8,9,10. We analyzed transmission of alpha-synuclein prionoids via peripheral routes in more detail and addressed the question whether misfolded alpha-synuclein may neuroinvade the CNS after a single intraglossal or intraperitoneal injection, a feature that previously had been shown for prions but not for misfolded alpha-synuclein. After injection of prions into the tongue, neuroinvasion of the CNS is achieved via propagation along the hypoglossal nerve of the tongue that leads to the nucleus of the hypoglossal nerve, which is located in the brain stem¹¹. As a mouse model we chose Tg(M83^+/-:Gfap-luc^+/-) mice that overexpress the A53T mutant of human alpha-synuclein from the prion promoter, and firefly luciferase under control of a Gfap promoter to monitor astrocytic activation by bioluminescence, as previously shown in the brain of prion-infected mice¹². In our hands bigenic Tg(M83^+/-:Gfap-luc^+/-) mice did not develop disease until 23 months of age as has been shown by others¹³. A single injection of human alpha-synuclein fibrils via the intraglossal or intraperitoneal route induced neurologic disease with pathology in the brains and spinal cords of Tg(M83^+/-:Gfap-luc^+/-) mice supporting the hypothesis that alpha-synuclein prionoids share important characteristics with prions¹⁴.

Protocol

All procedures including animals were performed with approval of the animal protection committee of North Rhine-Westphalia State Environment Agency (LANUV). Animals were housed and cared for according to standard conditions with a 12 h light/dark cycle and free access to food and water.

1. Animal Model

1. Intercross hemizygous Tg(Gfap-luc^+/-) mice with hemizygous Tg(M83^+/-) mice to generate hemizygous bigenic Tg(M83^+/-:Gfap-luc^+/-) mice^15,16.
2. Genotype the progeny with real-time PCR for the presence of the transgene encoding human alpha-synuclein and with standard PCR for firefly luciferase^12,17.
3. Inoculate the animals at an age of six-to-eight weeks.

2. Inoculum Preparation

1. Prepare monomeric recombinant human alpha-synuclein with the A53T mutation in Tris-buffered saline (TBS) buffer containing 150 mM NaCl in 20 mM Tris-HCl (pH 7.2) as has been previously described¹⁴.
2. Prepare fibrillar alpha-synuclein for inoculations by agitating 3 µg/µL of monomeric recombinant human alpha-synuclein in an orbital shaker at 800 rpm and 37 °C for 5 days.
3. Dilute fibril assemblies in phosphate-buffered saline (PBS) to reach a final concentration of 1 µg/µL for intraperitoneal or 2 µg/µL for intraglossal inoculations.
4. Fragment the alpha-synuclein fibrils with a rod sonicator for 1 min (40 pulses of 0.5 s duration with a one-second pause between each pulse and an amplitude set to 50%) on ice. To avoid contamination by cross-seeding. Use clean sonication probes to prepare different inoculum.

3. Intraglossal Injections

1. Anesthetize animals with an intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine. Pinch the animal's toe and confirm that it does not withdraw its hindlimb to ensure proper anesthesia. Use vet ointment on the animal's eyes to prevent dryness while under anesthesia.
2. Fix narcotized animals carefully with adhesive tape onto a heating plate in a dorsal recumbent position with their heads facing towards the investigator.
3. Fill a 27 G disposable hypodermic syringe with 5 µL of sonicated alpha-synuclein fibrils or PBS.
4. Use one blunt-nosed thumb forceps with serrated tips to hold the animal’s mouth open, and a second smaller pair of forceps to carefully pull out the tongue to make the bottom side of the tongue accessible for injection.
5. Inject the needle of the syringe into the right or left bottom side of the tongue in proximity to the hypoglossal nerve. Slowly inject the inoculum after 5 s. Slowly retract the needle after 5 s to ensure that the inoculum has penetrated the tissue and is not lost while retracting the needle.
6. Release the animal from its fixations and leave it on the heating plate under constant monitoring until complete recovery.

4. Intraperitoneal Injections

1. For intraperitoneal injections, narcotize the animals shortly in an anesthesia chamber by inhalation with isoflurane/oxygen using a flow rate of 2 L/min and the vaporizer set to 2%. Pinch the animal's toe and confirm that it does not withdraw its hindlimb to ensure proper anesthesia.
2. Fill a 27 G disposable hypodermic syringe with 50 µL of sonicated alpha-synuclein fibrils or PBS.
3. Directly inject into the peritoneum of the mouse and avoid penetrating the small intestine or cecum located behind the abdominal wall by holding the animal in a dorsal position with the head facing away from the investigator and downward at approximately 45°. Monitor animals until they have recovered from the anesthesia.

5. Bioluminescence Imaging

1. Image Tg(M83^+/-:Gfap-luc^+/-) mice every 2-4 weeks using a bioluminescence imaging system.
2. Prior to imaging shortly anesthetize mice in an isoflurane/oxygen chamber with a flow rate of 2 L/min and the vaporizer set to 2%. Pinch the animal's toe and confirm that it does not withdraw its hindlimb to ensure proper anesthesia.
3. Shave and depilate the heads of the mice with a depilatory cream. Color the ears in black with a non-irritating marker to block unspecific bioluminescence.
4. Dilute D-luciferin potassium salt, the substrate of luciferase, in PBS to a 30 mg/mL stock solution. Store this in 1 mL aliquots at -20 °C. Protect the dissolved D-luciferin from exposure to light.
5. Weigh the animals prior to injection. Calculate the exact volume of D-luciferin for injection of 150 mg/kg body weight. Intraperitoneally inject D-luciferin and return the animal to the anesthesia chamber.
6. Start the imaging software. After the imaging system has reached its operating temperature initialize the system by clicking on the 'Initialize' button in the control panel.
7. Select the buttons 'Luminescent' and 'Photograph'. Set the exposure time to 60 s, the binning to 'Medium', the F/Stop to '1', and the EM gain to 'Off'.
8. Confirm that the excitation filter is set to 'Block' and the emission filter to 'Open', and set the subject height to 1.50 cm.
9. Ten min after injection with D-luciferin, place the animals onto the heating plate in the imaging chamber and ensure that their muzzles are correctly placed in the anesthesia outlet. Close the isoflurane/oxygen flow from the inhalation chamber and open the flow for the imaging chamber with a flow rate of 0.25 L/min and an evaporation of 2%. Close the door of the imaging chamber properly.
10. Click on the 'Acquire' button to measure the bioluminescence, which takes 60 s. Stop the isoflurane flow and monitor the animals until they have completely recovered after returning them back to their cages.
11. Use the imaging software to quantify the bioluminescence. Within the 'Tool Palette' select 'ROI Tools'. Under 'Type' within the 'ROI Tools' select 'Measurement ROI'.
12. Within the 'ROI Tools' click the "circle" tool and select the number of ROIs to draw from the pull down menu – ideally '3' for three mice.
13. In the image, right-click each ROI and under 'Properties' adjust the width and height to 1.25 cm. Position each ROI over the brain area that is quantified.
14. In the upper left of the image, set the units panel to 'radiance (photons)'.
15. Within the 'Tool Palette', select 'Image Adjust' and adjust the minimum and the maximum of the 'Color Scale', for instance, from 0.20e6 to 1.00e6.
16. Under 'File' save the data with 'Save'.

6. Biochemical Analysis

1. Isolate the brains and spinal cords according to established protocols^18,19.
2. Homogenize whole brain and whole spinal cord samples separately in PBS in the presence of protease and phosphatase inhibitors (diluted to 1x in PBS) in two 30 s cycles with 6,000 rpm in a tissue homogenizer. Ensure that the brain and spinal cord samples are homogenized to a final concentration of 20% (wt/vol).
3. Sonicate the samples twice while keeping them on ice with a constant pulse of 10 s and an amplitude set to 50%.
4. Adjust the homogenates to 10% in PBS and 750 mM NaCl by diluting them 1:1 with 1.5 M NaCl in PBS.
5. To separate the cellular debris, centrifuge the homogenates at 1,000 x g for 5 min at 4 °C. Keep the supernatants for the next steps and discard the pellets.
6. Measure the protein concentration in the homogenates using the bicinchoninic acid (BCA) assay according to the manufacturer's instructions. Incubate 1.0 mg of total protein from brain homogenates or 0.8 mg from spinal cord homogenates in N-laurylsarcosyl at a final concentration of 10% (wt/vol) for 15 min on ice.
7. Overlay the homogenates onto a 3 mL sucrose cushion of 10% (wt/vol) in distilled water and ultracentrifuge them at 465,000 x g for 1 h at 4 °C.
8. Resuspend the pellets in a buffer containing 4% sodium dodecyl sulfate (SDS), 2% β-mercaptoethanol, 192 mM glycine, 25 mM Tris, and 5% (wt/vol) sucrose.
9. Boil the samples at 100 °C for 5 min and load them onto SDS-polyacrylamide gels for electrophoresis in a morpholineethanesulfonic acid (MES) buffer system.
10. Transfer the separated proteins to polyvinylidene difluoride (PVDF) membranes in a semidry blotting system.
11. Incubate the membranes in 0.4% (vol/vol) formalin solution in PBS for 30 min at room temperature on a rotor with 35 rpm to cross-link the proteins with the membrane.
12. Block the membranes in TBS buffer containing 0.05% (vol/vol) Tween 20 and 5% (wt/vol) milk for 1 h at room temperature.
13. Incubate the blots with the primary antibody in blocking buffer overnight at 4 °C.
14. Wash the membranes three times in TBS buffer and incubate them with peroxidase- or fluorophore-conjugated secondary antibodies in TBS for 1 h at room temperature.
15. Visualize the bound secondary antibodies by chemiluminescence or fluorescence according to established protocols²⁰.

7. Immunofluorescence Analysis

1. Dehydrate the dissected brains and spinal cords in a tissue processing station and embed them into paraffin using a paraffin station.
2. Cut the paraffin-embedded tissues with a microtome in 8 µm thick coronal sections and mount the sections on glass slides.
3. Deparaffinize the tissue sections by incubating them in two separate xylol baths for 5 to 10 min and rehydrate them through a series of graded ethanol baths (100%, 90%, 70%, 50%) and finally with H₂O.
4. Incubate the sections in 0.01 M citrate buffer (pH 6.0) for 5 min at room temperature and additionally boil them for 10 min in a microwave oven.
5. Let the sections cool down to room temperature and incubate them with a 3% hydrogen peroxide solution for 30 min to inhibit endogenous peroxidases.
6. Block the tissue sections by incubation with 20% (vol/vol) normal goat serum, 1% (vol/vol) bovine serum albumin (BSA), and 0.5% Triton X-100 in PBS for 1 h at room temperature.
7. Incubate the sections with the primary antibody diluted in 1% (vol/vol) normal goat serum, 1% (vol/vol) BSA, and 0.25% Triton X-100 in PBS overnight at 4 °C.
8. Wash the sections twice with 0.25% (vol/vol) Triton X-100 in PBS and once with PBS.
9. Stain the sections with the corresponding fluorophore-conjugated secondary antibodies and the nuclear dye DAPI diluted in 1% (vol/vol) normal goat serum, 1% (vol/vol) BSA, and PBS for 1 h at room temperature.
10. After washing twice with 0.25% (vol/vol) Triton X-100 in PBS and once with PBS, coverslip the slides with embedding media and visualize the staining with a confocal laser scanning microscope.

Results

Peripheral injection of alpha-synuclein prionoids via the tongue or the peritoneum induced neuropathology in the CNS of bigenic Tg(M83^+/-:Gfap-luc^+/-) mice (Table 1 and Figure 1). After a single intraperitoneal injection with alpha-synuclein fibrils, four of five mice developed neurologic disease with a median incubation time of 229 ±17 days. Surprisingly, only one of five mice developed CNS disease after intraglossal i...

Discussion

Peripheral injection of alpha-synuclein fibrils into the peritoneum of Tg(M83^+/-:Gfap-luc^+/-) mice represents a facile method to induce neurologic disease accompanied by neuroinflammation to recapitulate important characteristics of synucleinopathies. Similarly, tongue injection represents another route for neuroinvasion by alpha-synuclein prionoids in transgenic mice but is less efficient. We chose to terminate our experiments at 420 days after injection and we cannot exclude the possibili...

Materials

Name	Company	Catalog Number	Comments
anti-actin antibody	Merck Millipore	MAB1501
anti-alpha-synuclein, phospho S129 antibody [pSyn#64]	Wako	015-25191
anti-alpha-synuclein, phospho S129 antibody [EP1536Y]	Abcam	ab51253
anti-GFAP antibody	Dako	Z0334 01
anti-IBA-1 antibody	Wako	019-19741
anti-Sequestosome-1 (p62) antibody	Proteintech	18420-1-AP
anti-ubiquitin antibody [Ubi-1]	Merck Millipore	MAB1510
Phosphate-buffered saline (PBS)	Invitrogen	14190169
Ketamine	Ratiopharm		100 mg/kg
Xylazine	Ratiopharm		10 mg/kg
27 G syringe	VWR	613-4900
Isoflurane	Piramal Healthcare	PZN  4831850
Depilatory cream	Veet
Secureline lab marker	Neolab	25040
D-luciferin potassium salt	Acris	LK10000	30 mg/mL stock solution
Thermomixer	Eppendorf	5776671
Sonopuls Mini20 sonicator	Bandelin	3648
IVIS Lumina II imaging system	PerkinElmer
Living Image 3.0 Software	PerkinElmer
Tg(M83+/-) mice or B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J mice	The Jackson Laboratory	004479
Standard pattern forceps	Fine Science Tools	11000-16
Narrow pattern forceps	Fine Science Tools	11002-12
N-laurylasarcosyl	Sigma	L5125-100G
Optima Max-XP ultracentrifuge	Beckman Coulter		TLA-110 rotor
Thickwall polycarbonate tubes	Beckman Coulter	362305
NuPAG Novex 4-12% Bis-Tris Midi Protein Gels	Thermo Fisher Scientific	WG1401BOX
HRP conjugated antibody	Cayman	Cay10004301-1
IR Dye 680 conjugated antibody	LI-COR Biosciences	926-68070
SuperSignal West Dura Extended Duration Substrate	Thermo Fisher Scientific	34075
Stella 3200 imaging system	Raytest
Odyssey infrared imaging system	LI-COR Biosciences
Tween 20	MP Biomedicals	TWEEN201
Triton X-100	Sigma	SA/T8787
Immobilon-FL PVDF membrane	Merck Millipore	IPFL00010
Xylol	Sigma	Roth
Hydrogen peroxide	Sigma	SA/00216763/000500	working solution 3%
Bovine serum albumine (BSA)	Thermo Fisher Scientific	A3294-100G
Goat serum	Thermo Fisher Scientific	PCN5000
4,6-diamidino-2-phenylindole (DAPI)	Thermo Fisher Scientific	D1306	working dilution 1:50,000
Fluoromount media	Omnilab	SA/F4680/000025
LSM700 confocal laser scanning microscope	Carl Zeiss
HALT protease and phosphatase inhibitors	Thermo Fisher Scientific	10516495
Precellys 24-Dual homogenizer	Peqlab	91-PCS24D
Alexa Fluor 488 conjugated antibody	Thermo Fisher Scientific	A31619
Alexa Fluor 594 conjugated antibody	Thermo Fisher Scientific	A11005
Pierce BCA Protein Assay Kit	Thermo Fisher Scientific	10741395
Microtome RM2255	Leica
LSM700 confocal laser scanning microscope	Carl Zeiss