Technique for Intranasal Administration of α-Synuclein Aggregates

Summary

The present protocol describes a method for intranasal administration of α-synuclein aggregates. This method provides insights into α-synuclein propagation from the olfactory mucosa to the olfactory bulb in Parkinson's disease.

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of Lewy bodies, which are aggregates of α-synuclein (α-Syn). Recently, the disease was proposed to develop and progress through the prion-like propagation of α-Syn aggregates from the olfactory bulb (OB) or dorsal nucleus of the vagus nerve. Although the origin of α-Syn aggregates in the OB remains unclear, their propagation from the olfactory mucosa has been recently suggested. We previously showed that intranasal administration of α-Syn aggregates in a mouse model induced α-Syn pathology in the OB of mice. In this study, we present a method of intranasal administration of α-Syn aggregates that induced α-Syn pathology in the OB of mice. Intranasal administration of α-Syn aggregates is a very simple and straightforward method, and we believe it will be a useful tool in the research for elucidating the origin of α-Syn pathology in the OB and the pathway of α-Syn propagation through the olfactory system.

Introduction

Parkinson's disease (PD), which is characterized by motor symptoms such as bradykinesia, resting tremors, and muscle rigidity, is the second most common neurodegenerative disorder¹. PD also presents non-motor symptoms, including olfactory dysfunction, cognitive impairment, depression, hallucinations, constipation, and orthostatic hypotension. Its pathological hallmarks are the dopaminergic cell death in the substantia nigra and the presence of α-synuclein (α-Syn) aggregates, called Lewy bodies².

Of note, α-Syn is a 140-amino acid protein that exists in the form of a soluble monomer (or tetramer) under normal conditions. However, under abnormal conditions, the soluble monomer is converted to insoluble high-molecular-weight aggregates, including oligomers and fibrils. The transition of α-Syn into oligomers and fibrils is reportedly involved in cellular toxicity³.

Recent studies have suggested the prion-like propagation of α-Syn aggregates between neurons. Based on numerous postmortem examinations, Braak et al. proposed in 2003 the hypothesis that Lewy body pathology spreads progressively in the brain in a somewhat stereotypic manner (Braak's hypothesis)^4,5. In 2008, postmortem examination of patients with PD who received fetal midbrain transplants revealed Lewy bodies in dopaminergic neurons derived from fetal tissues^6,7. These studies suggested that α-Syn aggregates could spread from the diseased brain to grafts, supporting Braak's hypothesis.

Following these observations, experiments involving primary neuronal cultures and intracerebral injection of α-Syn aggregates in mice have reproduced the spreading of Lewy body-like aggregates, providing further evidence for α-Syn propagation in a prion-like manner^8,9.

Braak et al. showed that the Lewy body pathology in PD initiates in the olfactory bulb (OB) and/or the dorsal nucleus of the vagus nerve (dmX)⁴. Based on Braak's hypothesis, several studies have reported the administration of α-Syn aggregates or Lewy body extracts from PD brains into the OB and gastrointestinal tract of experimental animals^10,11,12. In 2018, a study demonstrated that the administration of α-Syn aggregates into the OB of wild-type mice induced the propagation of α-Syn pathology along the olfactory pathway, resulting in olfactory dysfunction¹³. We previously inoculated α-Syn aggregates into the OB of α-Syn transgenic mice and found that this led to hippocampal atrophy and memory impairment¹⁴.

In 2022, we inoculated α-Syn aggregates into the OB of marmosets, a small non-human primate; this resulted in the propagation of α-Syn pathology along the olfactory pathway, OB atrophy, and widespread cerebral glucose hypometabolism¹⁰.

However, if the propagation of α-Syn aggregates occurs from the OB, a critical question arises: by which mechanism do α-Syn aggregates initially appear? Saito et al. previously reported the presence of Lewy bodies in the nasal mucosa¹⁵. The presence of α-Syn aggregates was detected in the nasal mucosa of patients with PD and multiple system atrophy (MSA) using real-time quaking-induced conversion (RT-QUIC) analysis¹⁶. Notably, analyzing nasal mucosa samples from patients with rapid eye movement sleep behavior disorder (RBD), which is considered a prodromal stage of PD, revealed an increase in α-Syn levels¹⁷. This study suggested that α-Syn pathology might exist in the nasal mucosa even from the prodromal phase of PD.

While these findings suggested a potential route from the nasal mucosa to the OB, there has been limited experimental evidence supporting this scenario. To address this gap, we administered α-Syn aggregates into the nasal cavity of mice and investigated the propagation of α-Syn pathology from nasal mucosa to the OB. Our experimental approach demonstrated that a single-dose intranasal administration of α-Syn aggregates in wild-type mice induced α-Syn pathology in the OB, providing experimental evidence for the propagation pathway from the nasal mucosa to the OB.

Protocol

C57BL/6J male mice 2 months old were used for this study. All experimental procedures were performed according to national guidelines. The Animal Research Committee of Kyoto University granted ethical approval and permission for this study (MedKyo 23,544).

1. Intranasal administration of α-Syn preformed fibrils

1. Prepare mouse α-Syn fibrils solution in PBS (4 mg/mL) according to previously reported methods¹¹. Wrap the tube with a transparent film to prevent contamination.
2. Sonicate 200 µL of α-Syn fibrils solution to generate α-Syn preformed fibrils (PFFs) using a water bath-type sonicator (Table of Materials). Fill the ultrasonic bath with water and add ice cubes to cool down to 4 °C for sonication. Sonicate fibrils for a total of 5 min with each cycle consisting of 30 s of sonication followed by a 30 s interval (a total of five cycles).
   NOTE: Use personal protective equipment such as disposable gloves, mask, and safety goggles to prevent the exposure of α-Syn.
3. Prepare a P10 pipette (Table of Materials) with a 10 µL pipette tip. Anesthetize each mouse using a combination of medetomidine hydrochloride, midazolam, and butorphanol (MMB) by intraperitoneal (i.p.) injection. The MMB combination anesthetic agent contained midazolam (0.3 mg/kg), medetomidine (4 mg/kg), and butorphanol tartrate (5 mg/kg). Use 0.005 mL/g by i.p. injection when mixing midazolam (1 mg/mL) 0.3 mL, medetomidine (5 mg/mL) 0.8 mL, butorphanol tartrate (5 mg/mL) 1 mL, and saline 2.9 mL.
4. Wait for 10 min to ensure that mice were completely anesthetized. Proper anesthetization can be confirmed if the mouse is in lateral recumbency and cannot right itself. Once anesthetized, apply ophthalmic ointment by using a sterile cotton applicator to prevent drying of the eyes.
   NOTE: Without anesthesia, when administering nasal doses, it is difficult to keep the mice supine and to administer α-Syn solution properly as the mice sneeze. For these reasons, sedation was necessary.
5. Place the anesthetized mouse in the supine position. Place a paper towel or similar material under the body, ensuring a slightly downward tilt of the head (Figure 1A,B). This positioning prevents the administered α-Syn solution from quickly flowing into the lungs or esophagus, facilitating its retention in the nasal cavity. The nostrils are seen in Figure 1C.
6. Draw 1 µL of α-Syn solution (4 mg/mL) into a P10 pipette and place the pipette tip near the mouse's nose. Slowly form a round drop and keep it at the end of the pipette tip (Figure 1D, red arrow). Intranasal administration is performed for the unilateral nostril, use the contralateral side as the control side.
7. Bring the drop close to one side of the mouse nostrils and allow the mouse to inhale it naturally. Watch the inhalation and wait for 30 s to 1 min (Figure 1E).
8. Repeat steps 1.6.-1.7. until a total volume of 20 µL of α-Syn solution is administered. After all procedures, discard the gloves and wipe a bench with commercial detergents if the surface is contaminated with α-Syn
   NOTE: The entire procedure should be conducted in a safety cabinet to prevent the inhalation of α-Syn fibrils by the personnel performing the procedure. A previous report has shown that the volume of 25 µL is the most efficient for nose-to-brain drug administration¹⁸. We use a similar volume of 20 µL in the present study. We also use the same concentration of PFFs as that for injection into the OB in the previous report¹⁰, which is close to the 400 µM (5.6 mg/mL) of PFFs¹⁹.
9. Administer atipamezole (3 mg/kg; Table of Materials) by i.p. injection to reverse medetomidine and facilitate recovery. Use 0.005 mL/g by i.p. injection when mixing atipamezole (5 mg/mL) 0.6 mL and saline 4.4 mL.
   NOTE: Provide analgesia because it is not fully understood how stressful the intranasal administration of α-Syn would be.
10. Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency. After full recovery, return the mouse to the cage.

2. Sample preparation

1. Sacrifice the treated mice at 1, 3, 6, and 12 months after intranasal administration of α-Syn preformed fibrils.
   1. Put the mice into the induction box, administer sevoflurane (>6.5%) and continue until respiratory arrest occurs for > 60 s. Remove the mice and perform rapid exsanguination by right atrial incision to ensure euthanasia.
2. Insert a 25G needle (Table of Materials) into the apex of the heart. Perfuse the mouse at 4 mL/min with 15 mL of PBS and then 15 mL of 4% PFA in PBS.
3. Cut off the head with scissors and make a 2 cm incision in the scalp with a scalpel. Incise the cranial bone with scissors at the lateral side from the bottom to the nose (Figure 2A,B). To obtain the OBs, remove completely the cranial bone on the OBs (Figure 2B, yellow circle).
4. Turn over the head, sever the brain nerves, and then remove the brain carefully with forceps (Figure 2C, D). Obtain the brain with intact OBs (Figure 2E). To obtain the intact OBs, carefully cut the olfactory nerves with forceps as the OBs attach to the skull bone (Figure 2D, yellow circle).
5. Place the brain samples into the 4% PFA in PBS overnight at 4 °C. Do not leave the brain samples for more than 24 h.
   NOTE: Excessive fixation will decrease immunohistochemical staining. If long-term storage is required, preserve brain samples in 70% ethanol or PBS containing 0.1% sodium azide to maintain sterility.

3. Immunohistochemical staining of the OB

1. Paraffinize the samples using an automatic tissue processor (Table of Materials) as described below.
   1. Immerse in 70% ethanol for 120 min, followed by immersion in 100% ethanol for 8x for 60 min. Then, immerse in 100% xylene for 3x for 120 min.
   2. Immerse in paraffin at 60 °C for 120 min followed by immersion in paraffin at 60 °C for 240 min.
2. Embed the samples in paraffin as described below.
   1. Embed the samples in paraffin at 60 °C with a modular tissue embedding center (Table of Materials). Cool them on ice.
3. Cut the samples into 8 µm thick sections with a microtome (Table of Materials)¹⁸.
4. Perform deparaffinization as described below.
   1. Immerse in 100% xylene for 2x for 5 min, followed by immerse in 100% ethanol for 2x for 3 min.
   2. Immerse in 70% ethanol for 3 min. Wash in PBS for 3 min.
5. Conduct antigen retrieval as described below.
   1. Immerse in 100% formic acid for 15 min. Rinse in phosphate-buffered saline (PBS) for 2 min.
   2. Autoclave at 120 °C for 10 min. Allow it to cool naturally.
6. Immerse in 1% hydrogen peroxide in methanol for 10 min. Wash in PBS for 5 min.
7. Add 500 µL of 5% skim milk in PBS on the slides and incubate them for 30 min at room temperature.
8. Put the 500 µL of anti-phosphorylated α-Syn antibody (p-α-Syn, 1/10000) in PBS on the slides and incubate them overnight at 4 °C. Wash 2x with PBS for 5 min each.
9. Incubate with universal immuno-peroxidase polymer, anti-Rabbit (Table of Materials) for 1 h at 25 °C. Wash 2x with PBS for 5 min each.
10. Develop using the 3,3'-diaminobenzidine (DAB) staining kit (Table of Materials) for 5 min according to the manufacturer's instructions.
11. Immerse in hematoxylin solution for 1 min (Table of Materials). Decolorize in running water for 10 min.
12. Perform mounting as described below.
    1. Immerse in 70% ethanol for 5 min. Immerse in 100% ethanol for 2x for 5 min.
    2. Immerse in 100% xylene for 2x for 5 min. Mount using an embedding medium (Table of Materials). Apply the 100 µL of an embedding medium to the slides and cover with a glass coverslip.
13. Acquire images using an All-in-One microscope at 20x and 40x magnification (Table of Materials).

Results

Figure 3 shows several examples of α-Syn aggregates in the OB. In the present study, we administered α-Syn aggregates into the unilateral nostril. The two nasal cavities are separated by the nasal septum, and each OB projects the olfactory sensory neurons to each nasal cavity separately. Therefore, the OB on the contralateral side can be used as a control.

P-α-Syn pathology was not observed in the OB on the treated side after 1 and 3 months (

Discussion

In a previous study, the administration of α-Syn aggregates into the nasal cavity of macaques induced the death of dopaminergic cells and iron deposition in the substantia nigra, although α-Syn aggregates were not observed²¹. Daily administration of A53T human α-Syn aggregates into the nasal cavity of prion promoter α-Syn transgenic mice (M83 mice) for 28 days was reported to induce α-Syn pathology in the brains and motor symptoms in mice^19,

Materials

Name	Company	Catalog Number	Comments
All-in-One Fluorescence Microscope BZ-X710	KEYENCE	N/A	All-in-One microscope
Ampicillin Sodium Salt	Nacalai tesque	02739-32
Bioruptor II	Sonicbio	BR2006A	Water bath type sonicator.
Butorphanol tartrate	Meiji Seika Pharma	WAK-52850
Cellulose tube	MISUMI	UC20-32-100
DeepWellMaximizer	TAITEC	MBR-022UP	Shaker
DynaCompetent Cells Zip BL21(DE3)	BioDynamics Laboratory Inc.	DS255	Competent cell
Entellan	Sigma-Aldrich	107961	Rapid mounting medium for microscopy
Graefe Extra Fine Forceps Curved Serrated	FST	11152-10	forceps
Hardened Fine Scissors	FST	14090-09	scissors
Histofine Simple stain mouse MAX-PO (R)	Nichirei Bioscience	414341	Universal Immuno-peroxidase Polymer, anti-Rabbit
ImageJ ver 1.52p	NA	NA	https://imagej.net/
innova4200	New Brunswick scientific	9105085	Incubator shaker
Isopropyl-β-D-thiogalactopyranoside	Nacalai tesque	19742-94
LB broth, Lennox	Nacalai tesque	20066-24
Leica EG 1150 H	Leica	14 0388 86 108	Modular Tissue Embedding Center
Leica TP 1020	Leica	14 0422 85108	Automatic Tissue Processor
Medetomidine	Fuji Film	135-17473
Microm HM325 Rotary Microtome	Thermo Scientific	902100
Midazolam	Maruishi Seiyaku	4987-211-76210-0
New hematoxylin Type G	Muto	65-9197-38	Hematoxylin solution
Normal winged needle for vein D type, 25G	TERUMO	NN2332R	25G needle
Optima TLX Ultracentrifuge	Beckman Couler	8043-30-1197	Ultracentrifuge
P10 pipette	Gilson	FA10002P
Paraffin	Leica	39601095
paraformaldehyde	Nacalai tesque	30525-89-4
Peroxidase Stain DAB Kit	Nacalai tesque	25985-50
Pirece BCA Protein Assay Kits	Thermo Scientific	23225	BCA assay
pRK172	Addgene	#134504	Plasmid
Q-Sepharose Fast Flow.	cytiva	17051001	Ion exchange resin