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 ....

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 tra.......

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