PFF preparation tail (storage / sonication / pilot validation), Vargas 2019 Summary of a-syn animal models Table 1, Transgenic vs Vector vs Injection matrix, MOA of aSyn spreading axon transport table, Antibodies to aSyn start

PFF preparation (continued)

PFF storage / generation:

37°C with shaking for 7 days, and subjected to a quality test. α-Syn PFFs should not be stored at 4°C or -20°C because dissociation might occur. They should only be stored at -80°C.33 It is advisable to use freshly prepared PFFs. Samples stored in the shortterm at room temperature or at -80°C for up to 1.5 years retain pathogenicity but can lose activity.34 To generate pathogenic species of PFFs, the fibrils should be diluted and sonicated immediately before use. To achieve consistent pathogenicity, sonication must produce short fibrils with a length of 50 nm or shorter as the pathogenicity of α-syn PFFs depends on the structure and size of the fibrils.35,36 Other than regular quality control experiments used to validate the conversion of monomeric α-syn to PFFs, in vivo pilot studies are required to validate the pathogenicity of PFF species before any long-term in vivo studies. All parameters at every step in the standard protocol should be validated and tailored to each study’s purposes and models.34

Summary of a-syn animal models (J.Y. Vargas et al., Ageing Research Reviews 50 (2019) 89-101)

Table 1 — Summary of in vivo studies representing the major milestones in the understanding of α-syn prion-like spreading properties.

Experimental approach	Animals	Material assayed	Region of interest	Time points	References
Engrafted neurons on PD brains	Thy-1 α-syn transgenic mice	Mouse cortical neuronal stem cells	Hippocampus	4 weeks	Desplats et al., 2009
	Rats (WT) injected with AAV-α-syn	Rat embryonic ventral mesencephalic neurons	Striatum	1 week 2 weeks 4 weeks	Angot et al., 2012
Intracerebral inoculation/expression of α-syn	C57BL6/C3H mice (WT)	Mouse α-syn fibrils	Striatum	30 days 90 days 180 days	Luk et al., 2012a
	C57BL/6J mice (WT)	Human α-syn monomers and fibrils	Olfactory bulb	1 week, 3 h, 12 h, 72 h	Rey et al., 2013
	WT and α-syn null mice, monkeys	α-Syn extracts from the PD patients	Substantia nigra and striatum	4 weeks 16 weeks 56-68 weeks	Recasens et al., 2014
	Rats (WT)	Human α-syn oligomers, fibrils and ribbons. AAV-α-syn	Substantia nigra	7 days 120 days	Peelaerts et al., 2015
Peripheric administration of α-syn	WT and α-syn null mice	AAV-GFP AAV-α-syn	Vagus nerve	2 weeks 6 weeks 12 weeks	Helwig et al., 2016
	WT and α-syn transgenic mice	α-Syn fibrils	Sciatic nerve	14 weeks	Ayers et al., 2014b
	WT and α-syn transgenic mice	Gut microbiota from PD donors		12-13 weeks and 24-25 weeks	Sampson et al., 2016

Transgenic vs Vector-mediated α-syn vs Injection comparison

	NHP available?	a-syn expression level	a-syn accumulation/aggregation	a-syn propagation	Neurodegeneration	Behavior
Transgenic	X	Too much than human	o	X	X (alpha-synuclein cell loss has been reported in only 2 of the many mouse models over-expressing alpha-syn: a line expressing a doubly mutated protein, which is not found in patients, and in a line expressing a truncated alpha-syn [36, 37]; ↓ 50% reduction in TH-positive neurons, 24-weeks post-AAV injection)	X
Vector-mediated α-syn (vector-mediated α-syn overexpression)	O	Too much than human	O (inclusions develop)	X
Injection	o	Less than AAV	o	o	o

MOA of aSyn spreading

Retrograde axonal spread (transport) 보인다는 것 같음 (동물모델에서 sciatic nerve 까지 가는 듯).

de axons (showing direction)	Secreted into extracellular space	In ECF	Uptake	Recipient cell	Pathological aggregation	Lewy bodies	Total a-syn	Secondary secretion
retrograde AT (Brundt 2012)	Exosome		passive way (diffusion)	건강함 (Both fibrillar form of αSyn and non-fibrillar oligomeric αSyn and aggregated αSyn (in lysosome with a half life of 7 h in SH-SY5Y cells & RAT embryonic cortical neuron) [10] (Lee 2008 #620), monomeric αSyn 도 있지만 endogenous monomers αSyn 일컬 (Bae 2014 #610))	Co-aggregation with resident αSyn, can recruit other soluble monomers that assemble to form aggregates9	fiber but not like LB rounded
antero & retro AT (Brahic 2016)	exophagy		active way (endocytosis)	α-syn associates to lipid rafts, and more specifically to the glycolipid ganglioside GM1, which is abundantly expressed in the brain (Fortin et al., 2004; Olive et al., 1995).
	(Bae, 2014 #610) Aggregated aSyn 만 exocytosis 된다 b/c 이런 놈만 세포막에 구멍냄 ((Vasili, 2019 #616).	extracellular proteases, such as MMP3, directly cleaves secreted a-syn monomers
		Extracellular aSyn Activates microglia → kills DA neurons (Lee, 2008 #620)

Antibodies to aSyn (start)

	Pathologic conversion					Pathological aggregation
	monomeric	tetramer	Phosphorylated a-syn	Misfolded monomer	B-pleated sheet	Insoluble	Oligomeric (still soluble)	protofibril	insoluble amyloid-like fibril	Lewy bodies	Total a-syn
(antibody)			(20200414) ADx Neurosciences, TV Q3 FY2020, CS : Q42020, • 기존 : anti-pS129 a-syn antibody	(Waxman, 2008 #535) Syn505, Syn506 and Syn514 recognize conformational variants of α-syn	Thioflavin-staining (antibody는 아니지만)	(20200414) ADx Neurosciences, TV Q4 FY2020, CS : Q3 FY2021,	mouse anti-a-syn fibrils [02; [43]]