All experiments were supported by Rie Hikawa. We extend our thanks to Yasuko Matsuzawa for the paperwork. This study was supported by JSPS KAKENHI (M.S., No. JP19K23779, JP20K16493, and JP20H00663).

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 &#945;-Syn preformed fibrils
<ol>
	<li>Prepare mouse &#945;-Syn fibrils solution in PBS (4 mg/mL) according to previously reported methods11. Wrap the tube with a transparent film to prevent contamination.</li>
	<li>Sonicate 200 &#181;L of &#945;-Syn fibrils solution to generate &#945;-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 &#176;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 &#945;-Syn.</li>
	<li>Prepare a P10 pipette (Table of Materials) with a 10 &#181;L 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.</li>
	<li>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 &#945;-Syn solution properly as the mice sneeze. For these reasons, sedation was necessary.</li>
	<li>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 &#945;-Syn solution from quickly flowing into the lungs or esophagus, facilitating its retention in the nasal cavity. The nostrils are seen in Figure 1C.</li>
	<li>Draw 1 &#181;L of &#945;-Syn solution (4 mg/mL) into a P10 pipette and place the pipette tip near the mouse&#39;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.</li>
	<li>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).</li>
	<li>Repeat steps 1.6.-1.7. until a total volume of 20 &#181;L of &#945;-Syn solution is administered. After all procedures, discard the gloves and wipe a bench with commercial detergents if the surface is contaminated with &#945;-Syn 
	NOTE: The entire procedure should be conducted in a safety cabinet to prevent the inhalation of &#945;-Syn fibrils by the personnel performing the procedure. A previous report has shown that the volume of 25 &#181;L is the most efficient for nose-to-brain drug administration18. We use a similar volume of 20 &#181;L 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 &#181;M (5.6 mg/mL) of PFFs19.</li>
	<li>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 &#945;-Syn would be.</li>
	<li>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.</li>
</ol>
2. Sample preparation
<ol>
	<li>Sacrifice the treated mice at 1, 3, 6, and 12 months after intranasal administration of &#945;-Syn preformed fibrils.
	<ol>
		<li>Put the mice into the induction box, administer sevoflurane (&#62;6.5%) and continue until respiratory arrest occurs for &#62; 60 s. Remove the mice and perform rapid exsanguination by right atrial incision to ensure euthanasia.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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).</li>
	<li>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).</li>
	<li>Place the brain samples into the 4% PFA in PBS overnight at 4 &#176;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.</li>
</ol>
3. Immunohistochemical staining of the OB
<ol>
	<li>Paraffinize the samples using an automatic tissue processor (Table of Materials) as described below.
	<ol>
		<li>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.</li>
		<li>Immerse in paraffin at 60 &#176;C for 120 min followed by immersion in paraffin at 60 &#176;C for 240 min.</li>
	</ol>
	</li>
	<li>Embed the samples in paraffin as described below.
	<ol>
		<li>Embed the samples in paraffin at 60 &#176;C with a modular tissue embedding center (Table of Materials). Cool them on ice.</li>
	</ol>
	</li>
	<li>Cut the samples into 8 &#181;m thick sections with a microtome (Table of Materials)18.</li>
	<li>Perform deparaffinization as described below.
	<ol>
		<li>Immerse in 100% xylene for 2x for 5 min, followed by immerse in 100% ethanol for 2x for 3 min.</li>
		<li>Immerse in 70% ethanol for 3 min. Wash in PBS for 3 min.</li>
	</ol>
	</li>
	<li>Conduct antigen retrieval as described below.
	<ol>
		<li>Immerse in 100% formic acid for 15 min. Rinse in phosphate-buffered saline (PBS) for 2 min.</li>
		<li>Autoclave at 120 &#176;C for 10 min. Allow it to cool naturally.</li>
	</ol>
	</li>
	<li>Immerse in 1% hydrogen peroxide in methanol for 10 min. Wash in PBS for 5 min.</li>
	<li>Add 500 &#181;L of 5% skim milk in PBS on the slides and incubate them for 30 min at room temperature.</li>
	<li>Put the 500 &#181;L of anti-phosphorylated &#945;-Syn antibody (p-&#945;-Syn, 1/10000) in PBS on the slides and incubate them overnight at 4 &#176;C. Wash 2x with PBS for 5 min each.</li>
	<li>Incubate with universal immuno-peroxidase polymer, anti-Rabbit (Table of Materials) for 1 h at 25 &#176;C. Wash 2x with PBS for 5 min each.</li>
	<li>Develop using the 3,3&#39;-diaminobenzidine (DAB) staining kit (Table of Materials) for 5 min according to the manufacturer&#39;s instructions.</li>
	<li>Immerse in hematoxylin solution for 1 min (Table of Materials). Decolorize in running water for 10 min.</li>
	<li>Perform mounting as described below.
	<ol>
		<li>Immerse in 70% ethanol for 5 min. Immerse in 100% ethanol for 2x for 5 min.</li>
		<li>Immerse in 100% xylene for 2x for 5 min. Mount using an embedding medium (Table of Materials). Apply the 100 &#181;L of an embedding medium to the slides and cover with a glass coverslip.</li>
	</ol>
	</li>
	<li>Acquire images using an All-in-One microscope at 20x and 40x magnification (Table of Materials).</li>
</ol>

&#945;-Synuclein Propagation: Intranasal Administration in Parkinson&#39;s Disease

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+administration+of+&#945;-synuclein+preformed+fibrils+triggers+microglial+iron+deposition+in+the+substantia+nigra+of+Macaca+fascicularis.">Intranasal administration of &#945;-synuclein preformed fibrils triggers microglial iron deposition in the substantia nigra of Macaca fascicularis.</a> Cell Death Dis. 12 (1), 81 (2021).</li><li>Macdonald, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+&#945;-synuclein+and+neurodegeneration+in+the+central+nervous+system+of+heterozygous+M83+mice+following+the+peripheral+administration+of+&#945;-synuclein+seeds.">Assembly of &#945;-synuclein and neurodegeneration in the central nervous system of heterozygous M83 mice following the peripheral administration of &#945;-synuclein seeds.</a> Acta Neuropathol Commun. 9 (1), 189 (2021).</li><li>Abdelmotilib, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;-Synuclein+fibril-induced+inclusion+spread+in+rats+and+mice+correlates+with+dopaminergic+Neurodegeneration.">&#945;-Synuclein fibril-induced inclusion spread in rats and mice correlates with dopaminergic Neurodegeneration.</a> Neurobiol Dis. 105, 84-98 (2017).</li><li>Tarutani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+fragmented+pathogenic+&#945;-Synuclein+seeds+on+prion-like+propagation.">The effect of fragmented pathogenic &#945;-Synuclein seeds on prion-like propagation.</a> J Biol Chem. 291 (36), 18675-18688 (2016).</li><li>Taguchi, T., Ikuno, M., Yamakado, H., Takahashi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+model+for+prodromal+Parkinson's+disease.">Animal model for prodromal Parkinson's disease.</a> Int J Mol Sci. 21 (6), 1961 (2020).</li><li>Bousset, L., Brundin, P., B&#246;ckmann, A., Meier, B., Melki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+procedure+for+removal+and+inactivation+of+alpha-Synuclein+assemblies+from+laboratory+materials.">An efficient procedure for removal and inactivation of alpha-Synuclein assemblies from laboratory materials.</a> J Parkinsons Dis. 6 (1), 143-151 (2016).</li><li>Fenyi, A., Coens, A., Bellande, T., Melki, R., Bousset, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+efficacy+of+different+procedures+that+remove+and+disassemble+alpha-synuclein,+tau+and+A-beta+fibrils+from+laboratory+material+and+surfaces.">Assessment of the efficacy of different procedures that remove and disassemble alpha-synuclein, tau and A-beta fibrils from laboratory material and surfaces.</a> Sci Rep. 8, 10788 (2018).</li></ol>

The authors have no conflicts of interest to disclose.

In a previous study, the administration of &#945;-Syn aggregates into the nasal cavity of macaques induced the death of dopaminergic cells and iron deposition in the substantia nigra, although &#945;-Syn aggregates were not observed21. Daily administration of A53T human &#945;-Syn aggregates into the nasal cavity of prion promoter &#945;-Syn transgenic mice (M83 mice) for 28 days was reported to induce &#945;-Syn pathology in the brains and motor symptoms in mice19,22. In the present study, we demonstrated that a single-dose administration of &#945;-Syn aggregates into the nasal cavity of wild-type mice induced &#945;-Syn pathology in the OB. This simple method could be considered valuable for investigating &#945;-Syn propagation from the nasal mucosa to the OB.
Sufficient sonication of &#945;-Syn fibrils in this method is crucial because previous reports showed the small size of fragmented &#945;-Syn aggregates is the most efficient for triggering &#945;-Syn pathology23,24. Insufficient sonication of &#945;-Syn fibrils will result in little propagation to the OB. As different sonicators have varying sonication power, optimization of this process is necessary. The supine posture of the mouse when the &#945;-Syn solution is administered into the mouse's nostrils is also crucial. If the mouse's head is elevated, the &#945;-Syn solution will flow into the lungs and esophagus. Inappropriate posture can cause unexpected death or insufficient &#945;-Syn propagation.
In the present study, i.p. used an MMB combination anesthetic agent for anesthesia. MMB combination anesthesia was reported to provide good sedation and analgesia, and its effect could be rapidly reversed by atipamezole23. Since inhalational anesthesia requires covering the nostrils with a mask to maintain the anesthesia, it is more difficult to administer the &#945;-Syn solution intranasally. Additionally, the rotenone model has been studied widely and is a very useful model25. However, it is a drug-induced model and may have different pathomechanisms than idiopathic Parkinson's disease (iPD)25. Since this model induced &#945;-Syn propagation, we believe it is closer to the iPD. 
Our study also suggested that exogenous &#945;-Syn aggregates could transmit through the nasal mucosa. Therefore, when researchers are conducting experiments using &#945;-Syn aggregates, precautions should be taken to avoid the inhalation of &#945;-Syn aggregates. For instance, &#945;-Syn aerosols could be generated during the sonication step. We recommend the use of a sealed type sonicator or sonication within a safety cabinet. Additionally, mice may sneeze, potentially generating &#945;-Syn aerosols. To mitigate this risk, all operations should be performed within a safety cabinet. Also, researchers should use personal protective equipment such as disposable gloves, masks, and safety goggles to prevent exposure to &#945;-Syn. After all procedures, researchers need to wipe a bench with commercial detergents if the surface is contaminated with &#945;-Syn. Previous reports have shown that both commercial detergents and SDS detached aggregates from contaminated surfaces26,27.
In this study, paraffin sections were used to evaluate &#945;-Syn pathology in the OB, as the small structure of the OB is better preserved in paraffin sections compared with that in frozen sections. This study showed that &#945;-Syn pathology was induced in the OB; however, the observed &#945;-Syn pathology was mild and limited and was mostly represented by thread-like aggregates known as Lewy neurites in the glomeruli. 
It should be noted that apparent behavioral changes in experimental mice were not observed in the present study. The &#945;-Syn pathology induced by this method may be insufficient to induce changes in the behavior phenotype. Further investigations are needed to elucidate the specific conditions leading to severe pathology and behavioral changes. In the future, if we could establish a more efficient way for propagation from the nasal mucosa to the OB, this simple method provides an alternative way to evaluate &#945;-Syn propagation without surgery technique.
 In addition, we used the OB on the contralateral side in the treated mice as a control. However, retrograde propagation from the anterior olfactory nucleus or piriform cortex to the contralateral OB could occur in a longer-term follow-up. In that case, non-treated mice were used as a control.
While this simple method provides valuable insights into &#945;-Syn propagation from the nasal mucosa to the OB, further studies are needed to comprehensively understand the mechanisms underlying PD.

Parkinson&#39;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 &#945;-synuclein (&#945;-Syn) aggregates, called Lewy bodies2.
Of note, &#945;-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 &#945;-Syn into oligomers and fibrils is reportedly involved in cellular toxicity3.
Recent studies have suggested the prion-like propagation of &#945;-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&#39;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 tissues6,7. These studies suggested that &#945;-Syn aggregates could spread from the diseased brain to grafts, supporting Braak&#39;s hypothesis.
Following these observations, experiments involving primary neuronal cultures and intracerebral injection of &#945;-Syn aggregates in mice have reproduced the spreading of Lewy body-like aggregates, providing further evidence for &#945;-Syn propagation in a prion-like manner8,9.
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&#39;s hypothesis, several studies have reported the administration of &#945;-Syn aggregates or Lewy body extracts from PD brains into the OB and gastrointestinal tract of experimental animals10,11,12. In 2018, a study demonstrated that the administration of &#945;-Syn aggregates into the OB of wild-type mice induced the propagation of &#945;-Syn pathology along the olfactory pathway, resulting in olfactory dysfunction13. We previously inoculated &#945;-Syn aggregates into the OB of &#945;-Syn transgenic mice and found that this led to hippocampal atrophy and memory impairment14.
In 2022, we inoculated &#945;-Syn aggregates into the OB of marmosets, a small non-human primate; this resulted in the propagation of &#945;-Syn pathology along the olfactory pathway, OB atrophy, and widespread cerebral glucose hypometabolism10.
However, if the propagation of &#945;-Syn aggregates occurs from the OB, a critical question arises: by which mechanism do &#945;-Syn aggregates initially appear? Saito et al. previously reported the presence of Lewy bodies in the nasal mucosa15. The presence of &#945;-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 &#945;-Syn levels17. This study suggested that &#945;-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 &#945;-Syn aggregates into the nasal cavity of mice and investigated the propagation of &#945;-Syn pathology from nasal mucosa to the OB. Our experimental approach demonstrated that a single-dose intranasal administration of &#945;-Syn aggregates in wild-type mice induced &#945;-Syn pathology in the OB, providing experimental evidence for the propagation pathway from the nasal mucosa to the OB.

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

technique for intranasal administration of &#945;-synuclein aggregates

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of Lewy bodies, which are aggregates of &#945;-synuclein (&#945;-Syn). Recently, the disease was proposed to develop and progress through the prion-like propagation of &#945;-Syn aggregates from the olfactory bulb (OB) or dorsal nucleus of the vagus nerve. Although the origin of &#945;-Syn aggregates in the OB remains unclear, their propagation from the olfactory mucosa has been recently suggested. We previously showed that intranasal administration of &#945;-Syn aggregates in a mouse model induced &#945;-Syn pathology in the OB of mice. In this study, we present a method of intranasal administration of &#945;-Syn aggregates that induced &#945;-Syn pathology in the OB of mice. Intranasal administration of &#945;-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 &#945;-Syn pathology in the OB and the pathway of &#945;-Syn propagation through the olfactory system.

Figure 3&#160;shows several examples of &#945;-Syn aggregates in the OB. In the present study, we administered &#945;-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-&#945;-Syn pathology was not observed in the OB on the treated side after 1 and 3 months (Figure 3A, B). Lewy neurite-like p-&#945;-Syn aggregates were observed in the glomeruli on the treated side after 6 and 12 months. (Figure 3C,D,F,G). These aggregates were not observed in the OB on the control side (Figure 3E).
We can estimate the &#945;-Syn pathology by counting &#945;-Syn aggregates or examining the size, etc., by using ImageJ (Table of Materials). For example, we previously counted and reported the number of &#945;-Syn aggregates in this model20. In the OB on the treated side, Lewy neurite-like aggregates in the OB significantly increased over 12 months (one-way ANOVA, followed by Dunnett&#39;s test, p &#60; 0.01; Figure 3H). In the OB on the control side, there are few &#945;-Syn aggregates.
A previous study showed that injected PFFs into the mouse brain were degraded within a week, and endogenous mouse &#945;-Syn was converted into &#945;-Syn aggregates19. In the present study, the increase in &#945;-Syn pathology over time indicates that endogenous mouse &#945;-Syn has been converted to &#945;-Syn aggregates by single-dose intranasal administration of PFFs. These results indicated that &#945;-Syn pathology could spread from the olfactory mucosa to the OB.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66943/66943fig01.jpg" /> 
Figure 1: Mouse nasal cavity administration method. (A) The mouse is positioned in the supine position with the head slightly tilted downward. (B) The nostrils of the mouse are indicated by yellow arrowheads. (C, D) The &#945;-Syn fibril solution is dropped into the nostril of the mouse (C, red arrow) and quickly inhaled (D, red arrows). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66943/66943fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66943/66943fig02.jpg" /> 
Figure 2: Outline of steps to obtain the intact olfactory bulbs. (A) Using scissors, incise the cranial bone sagittally and laterally (yellow dash line). (B) Remove the cranial bone on the olfactory bulbs (OBs) completely. (C) Turn over the head and carefully remove the brain. Sever the brain nerves. (D) Cut the olfactory nerves attached to the cranial bone. (E) The whole brain with the intact OBs. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66943/66943fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66943/66943fig03.jpg" /> 
Figure 3: P-&#945;-Syn aggregates in glomeruli of the olfactory bulb. (A-G) P -&#945;-Syn (EP1536Y) immunohistochemistry of the OB on (A-D) the treated side 1, 3, 6, and 12 months (1 m, 3 m, 6 m, and 12 m, respectively) after the intranasal administration and (E) the control side 12 months after the intranasal administration. (C, D) Lewy neurite-like thread-like &#945;-Syn aggregates appear around the glomeruli. (F, G) Enlarged images are high-magnification images of Lewy neurite-like p-&#945;-Syn 6 and 12 months after the intranasal administration. (H) The amount of Lewy neurite-like p-&#945;-Syn deposition in the OB on the treated and control sides (n = 4-5). Graphs show mean &#177; SEM. One-way ANOVA followed by Dunnett&#39;s test; **&#60;0.01.&#160;This figure has been modified from a previous report20.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66943/66943fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Technique for Intranasal Administration of &#945;-Synuclein Aggregates

The present protocol describes a method for intranasal administration of &#945;-synuclein aggregates. This method provides insights into &#945;-synuclein propagation from the olfactory mucosa to the olfactory bulb in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of Lewy bodies, which are aggregates of &#945;-synuclein ...

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349 Views.  Kyoto University. 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.

Video: Technique for Intranasal Administration of α-Synuclein Aggregates

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Medicine

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

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

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 &#177;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.

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.

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

Immunology and Infection

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...

Compound Administration III

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
There are many commonly used routes for compound administration in laboratory mice and rats. However, certain protocols may require the use of less commonly used routes, including intradermal, intranasal, and intracranial injections. Specialized training is essential for these procedures to be performed successfully. Justification for these routes may need to be provided to gain Institutional Animal Care and Use Committee (IACUC) approval.

Compound Administration in Rodents- Intradermal, Intranasal &amp; Intracranial Routes

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
There are many commonly used routes for compound ...

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Immunostaining of Neurons Treated with Alpha-Synuclein Aggregates

Source: Panattoni, G., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57884">Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation.</a> J. Vis. Exp. (2018)
This video demonstrates the immunostaining of neurons treated with alpha-synuclein (&#945;S) aggregates. The neurons are first treated with &#945;S aggregates. They are then fixed, permeabilized, and treated with a blocking solution. Primary antibodies are added, followed by a fluorescently labeled secondary antibodies and a nuclear stain. The sample is then mounted and observed under a microscope.

Technique for Intranasal Administration of α-Synuclein Aggregates

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