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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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=Acceleration+of+oligomerization,+not+fibrillization,+is+a+shared+property+of+both+alpha-synuclein+mutations+linked+to+early-onset+Parkinson's+disease:+implications+for+pathogenesis+and+therapy.">Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson's disease: implications for pathogenesis and therapy.</a> Proc Natl Acad Sci U S A. 97 (2), 571-576 (2000).</li><li>Braak, 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=Staging+of+brain+pathology+related+to+sporadic+Parkinson's+disease.">Staging of brain pathology related to sporadic Parkinson's disease.</a> Neurobiol Aging. 24 (2), 197-211 (2003).</li><li>Braak, H., R&#252;b, U., Gai, W. P., Del Tredici, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+Parkinson's+disease:+possible+routes+by+which+vulnerable+neuronal+types+may+be+subject+to+neuroinvasion+by+an+unknown+pathogen.">Idiopathic Parkinson's disease: possible routes by which vulnerable neuronal types may be subject to neuroinvasion by an unknown pathogen.</a> J Neural Transm (Vienna). 110 (5), 517-536 (2003).</li><li>Kordower, J. H., Chu, Y., Hauser, R. A., Freeman, T., Olanow, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lewy+body-like+pathology+in+long-term+embryonic+nigral+transplants+in+Parkinson's+disease.">Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson's disease.</a> Nat Med. 14 (5), 504-506 (2008).</li><li>Li, J. Y., 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=Lewy+bodies+in+grafted+neurons+in+subjects+with+Parkinson's+disease+suggest+host-to-graft+disease+propagation.">Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host-to-graft disease propagation.</a> Nat Med. 14 (5), 501-503 (2008).</li><li>Luk, K. C., 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=Pathological+&#945;-synuclein+transmission+initiates+Parkinson-like+neurodegeneration+in+nontransgenic+mice.">Pathological &#945;-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice.</a> Science. 338, 949-953 (2012).</li><li>Desplats, P., 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=Inclusion+formation+and+neuronal+cell+death+through+neuron-to-neuron+transmission+of+alpha-synuclein.">Inclusion formation and neuronal cell death through neuron-to-neuron transmission of alpha-synuclein.</a> Proc Natl Acad Sci U S A. 106 (31), 13010-13015 (2009).</li><li>Sawamura, M., 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=Lewy+body+disease+primate+model+with+&#945;-Synuclein+propagation+from+the+olfactory+bulb.">Lewy body disease primate model with &#945;-Synuclein propagation from the olfactory bulb.</a> Mov Disord. 37 (10), 2033-2044 (2022).</li><li>Uemura, N., 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=Inoculation+of+&#945;-synuclein+preformed+fibrils+into+the+mouse+gastrointestinal+tract+induces+Lewy+body-like+aggregates+in+the+brainstem+via+the+vagus+nerve.">Inoculation of &#945;-synuclein preformed fibrils into the mouse gastrointestinal tract induces Lewy body-like aggregates in the brainstem via the vagus nerve.</a> Mol Neurodegener. 13 (1), 21 (2018).</li><li>Rey, N. L., 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=Widespread+transneuronal+propagation+of+alpha-synucleinopathy+triggered+in+olfactory+bulb+mimics+prodromal+Parkinson's+disease.">Widespread transneuronal propagation of alpha-synucleinopathy triggered in olfactory bulb mimics prodromal Parkinson's disease.</a> J Exp Med. 213 (9), 1759-1778 (2016).</li><li>Rey, N. L., 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=Spread+of+aggregates+after+olfactory+bulb+injection+of+&#945;-synuclein+fibrils+is+associated+with+early+neuronal+loss+and+is+reduced+long+term.">Spread of aggregates after olfactory bulb injection of &#945;-synuclein fibrils is associated with early neuronal loss and is reduced long term.</a> Acta Neuropathol. 135 (1), 65-83 (2018).</li><li>Uemura, N., 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+spread+from+olfactory+bulb+causes+hyposmia,+anxiety,+and+memory+loss+in+BAC-SNCA+mice.">&#945;-Synuclein spread from olfactory bulb causes hyposmia, anxiety, and memory loss in BAC-SNCA mice.</a> Mov Disord. 36 (9), 2036-2047 (2021).</li><li>Saito, Y., 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=Lewy+body+pathology+involves+the+olfactory+cells+in+Parkinson's+disease+and+related+disorders.">Lewy body pathology involves the olfactory cells in Parkinson's disease and related disorders.</a> Mov Disord. 31 (1), 135-138 (2016).</li><li>De Luca, C. M. G., 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=Efficient+RT-QuIC+seeding+activity+for+&#945;-synuclein+in+olfactory+mucosa+samples+of+patients+with+Parkinson's+disease+and+multiple+system+atrophy.">Efficient RT-QuIC seeding activity for &#945;-synuclein in olfactory mucosa samples of patients with Parkinson's disease and multiple system atrophy.</a> Transl Neurodegener. 8, 24 (2019).</li><li>Stefani, 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=Alpha-synuclein+seeds+in+olfactory+mucosa+of+patients+with+isolated+REM+sleep+behaviour+disorder.">Alpha-synuclein seeds in olfactory mucosa of patients with isolated REM sleep behaviour disorder.</a> Brain. 144 (4), 1118-1126 (2021).</li><li>Ucciferri, C. C., 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=Scoring+central+nervous+system+inflammation,+demyelination,+and+axon+injury+in+experimental+autoimmune+encephalomyelitis.">Scoring central nervous system inflammation, demyelination, and axon injury in experimental autoimmune encephalomyelitis.</a> J Vis Exp. (204), e65738 (2024).</li><li>Masuda-Suzukake, M., 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=Prion-like+spreading+of+pathological+&#945;-synuclein+in+brain.">Prion-like spreading of pathological &#945;-synuclein in brain.</a> Brain. 136 (Pt 4), 1128-1138 (2013).</li><li>Sawamura, M., 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=Single-dose+intranasal+administration+of+&#945;-syn+PFFs+induce+lewy+neurite-like+pathology+in+olfactory+bulbs.">Single-dose intranasal administration of &#945;-syn PFFs induce lewy neurite-like pathology in olfactory bulbs.</a> Parkinsonism Relat Disord. 112, 105440 (2023).</li><li>Guo, J. 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,</s...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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-&#946;-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&#160;</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.&#160;</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 (<s...

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 ...

technique-for-intranasal-administration-of-synuclein-aggregates

Research

JoVE Journal

Neuroscience

313 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

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. 
The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro9,5. Last, and perhaps most importantly, a small applied volume (&tilde;50&mu;l) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach1,8.

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. 
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most neuroimaging or neurophysiological investigations in humans have been confined to the study of &#947;-Amino-Butyric-Acid-(GABA)-receptor-agonist-based anesthetics, while the effects of dissociative N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthetics ketamine, nitrous oxide (N2O) and xenon (Xe) are largely unknown. This paper describes the methods underlying the simultaneous recording of magnetoencephalography (MEG) and electroencephalography (EEG) from healthy males during inhalation of the gaseous anesthetic agents N2O and Xe. Combining MEG and EEG data enables the assessment of electromagnetic brain activity during anesthesia at high temporal, and moderate spatial, resolution. Here we describe a detailed protocol, refined over multiple recording sessions, that includes subject recruitment, anesthesia equipment setup in the MEG scanner room, data collection and basic data analysis. In this protocol each participant is exposed to varying levels of Xe and N2O in a repeated measures cross-over design. Following relevant baseline recordings participants are exposed to step-wise increasing inspired concentrations of Xe and N2O of 8, 16, 24 and 42%, and 16, 32 and 47% respectively, during which their level of responsiveness is tracked with an auditory continuous performance task (aCPT). Results are presented for a number of recordings to highlight the sensor-level properties of the raw data, the spectral topography, the minimization of head movements, and the unequivocal level dependent effects on the auditory evoked responses. This paradigm describes a general approach to the recording of electromagnetic signals associated with the action of different kinds of gaseous anesthetics, which can be readily adapted to be used with volatile and intravenous anesthetic agents. It is expected that the method outlined can contribute to the understanding of the macro-scale mechanisms of anesthesia by enabling methodological extensions involving source space imaging and functional network analysis.

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

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 ...