Non-Spreading Models continued (WT Wade-Martins / F28tg / mjff / Mutant M83 PrP / KI rat / Truncated), Spreading Models, in vivo evidence, Atuka NHP results matrix

Animal models of aSyn — Non-Spreading Models (continued)

	line	Promoter	Human SNCA gene	Rodent SNCA gene
WT	Wade-Martins B6.Cg-Tg(SNCA)OVX37Rwm Sncatm1Rosl/J, JAX 023837		- Entire human SNCA gene (driven by the human SNCA promoter). Line was crossed with Snca KO to remove endogenous murine aSyn. Single integration site near centromere of Chr4. - ~2-fold overexpression of human aSyn with age-dependent striatal neurotransmission deficits, loss of nigral DA neurons, and motor dysfunction. - No concerns about genetic drift as line was cryorecovered in 2021 from sperm banked during deposit. - Industry licenses not required.	- No PD-related phenotypes reported. - Caveats: Industry licenses required with UCSF. Bred HOMxHOM at JAX for probably 10 years (expect genetic drift).
WT	F28tg mouse =F28Pka mouse		overexpresses human αSyn under the mouse Snca promotor and was created on a C57BL/6J background via pronuclear injection of a DNA sequence encoding human αSyn,
WT (일컬)	human aSyn w/o endog mouse aSyn (mjff)		'MJFF tools consortium 2022 Q3': failed
Mutant	PrP-A53T-SNCA TG (line M83), murine PrP (prion protein) promoter → Human A53T aSyn is expressed in neurons (oligodendrocyte 아니니까 MSA 에 부적합 모델임)		[SNCA Tg mice] This mouse contains the full human intronic, exonic, 5' and 3' UTR α-syn sequence on a null mouse α-syn background (Jax stock number 01079912). Expression of human α-syn transcript is ~8 times higher and human protein 1.3-2-fold higher vs endogenous mouse α-syn (SNCA ASO PRC) develops a severe and complex motor impairment, leading to paralysis and death, and pathologically showed Lewy body-like inclusions [9, 19]. → synaptic inhibition, Behavioral alteration But no significant nigrostriatal degeneration A53T-SNCA mice exhibit lower gba activity (~80% residual activity) (Rockenstein et al. 2016, PMID 27126635) Cortical lysates of PrP-A53T-SNCA mice have decreased gba activity (30), similar to that observed in the brains of sporadic PD patients expressing WT GBA1 alleles (32,33) (Rockenstein et al. 2016, PMID 27126635) ↓ GBA activity (~90% WT) (Rockenstein et al. 2016, PMID 27126635) (Emmanouilidou, 2011 #2600) fig3. ↑ aSyn (full length, monomer) in ISF	[dbl-PAC (SNCAA53T,Snca-/-) host] homozygous mutant transgene on a homozygous Snca knockout background (Snca murine protein). Prevail used this mice.
Mutant KI	A53T-SNCA Rat Rat		[Tools Consortium Co-Lead: Sean Clark, Amicus] Considering the conserved role for α-syn in genetic and idiopathic PD, its status as an attractive target for disease modifying therapies, and the fact that previous efforts to create α-syn KO and KI rat models were unsuccessful, the Tools Consortium funded a project to generate these novel rat models via a new targeting strategy utilizing the CRISPR-Cas9 system. This project was initiated in the Summer 2014 in partnership with Horizon Discovery (previously SAGE Labs). This project led to the creation of an α-syn KO model and an α-syn A53T KI model that expresses the humanized A53T α-syn protein without endogenous rat α-syn. The α-syn KO (strain ID 10150) and α-syn A53T KI (strain ID 10220) are now available for orders. Project Status: Complete; the lines are available at Horizon Discovery. Model phenotyping is ongoing (see below).
Truncated	MI2, tyrosine hydroxylase promoter		- expressing human, aggregation-prone truncated 1-120 α-syn under the tyrosine hydroxylase promoter. - alterations in gait impairment can be detected by the DigiGait test from 9 months of age, while gross motor deficit was detected by rotarod test at 20 months of age when 50% of dopaminergic neurons in the substantia nigra pars compacta are lost. - Synaptic dysfunction

The injection of human mutant α-syn by AAVs → a progressive, age-dependent loss of DA neurons, motor impairment, α-syn cytoplasmic inclusions, and degenerative changes in striatal axons both in rats [110, 111] and mice (Suzuki, 2020 #1029) observed in patients’ brains.

Spreading Models

Methods

Recombinant soluble a-syn proteins purified from bacterial cells, form amyloid-like fibrils that are morphologically and physicochemically similar to those observed in patients’ brains (Suzuki, 2020 #1029)

in vivo evidence

		Injection material	Recipient Species	Inoculation site	Observed when	Results	Mechanism implication
	(Desplats et al., 2009).	mouse cortical neuronal stem cells	TG mice expressing human a-syn	Hippocampus	1 m	15% of the engrafted cells were positive for human α-syn,
host → graft	Hansen et al., 2011).	mouse dopaminergic neurons	mice overexpressing human α-syn	striatum	6m	the transfer of α-syn
	(Bae, 2014 #610)	SH-SY5Y cells	mice overex-pressing human α-syn			engrafted cells were positive for human α-syn, 반면 endogenous aSyn은 미검출됨.	Seed 가 돌아다니고 aggregation 증가하네.
	(Kordower et al., 2008; Li et al., 2008)	embryonic neurons	PD patients	brain		LB-like pathology in the engrafted tissue
		Aggregared a-syn	WT mice			Spreading of a-syn in the CNS of injected mice
			TG mice overexpressing human WT a-syn			Spreading of a-syn in the CNS of injected mice
			TG mice overexpressing human mutated a-syn (A53T)			Spreading of a-syn in the CNS of injected mice
	(Karampetsou et al. 2017 Sci Reports).	Phosphorylation of aSYN in PFF at Ser129				significantly potentiates the associated pathology and promotes seeding (
	(Mason et al., 2016).	α-syn fibrils	non-transgenic mice,	OB	three months	α-syn positive inclusions in the temporary lobe (in the piriform and entorhinal cortices, amygdala and the hippocampus)
		α-syn of human	mice			a-syn positive inclusion (?) both human and mouse recombinant α-syn fibrils are able to efficiently induce α-syn pathology in injected mice, but others have reported the existence of a cross-seeding species barrier for human and mouse α-syn.
	(Sorrentino et al., 2017).	α-syn fibril	α-syn-transgenic and non-transgenic mice	striatum		α-syn spreading takes place in regions that are not anatomically connected	Other besides synaptic spreading: exogenous human asyn was actively internalized within host neurons and triggered the pathological conversion of endogenous asyn.
	(Recasens, 2014 #1248)	α-syn-containing extracts from the SN of PD patients, (주: PFF 아님!)	mice	SN		At the onset of LB-induced degeneration, host pathological α-syn diffusely accumulated within nigral neurons and anatomically interconnected regions, both anterogradely and retrogradely. LB-induced pathogenic effects required both human α-syn present in LB extracts and host expression of α-syn.
			macaque monkeys	striatum o		marked ↓ DA neurons in the SNpc and a widespread distribution of α-syn pathology (putamen, SNpc, globus pallidus, precentral gyrus, superior frontal gyrus, and entorhinal area in the temporal cortex (14 m after injection); trigger the aggregation of endogenous a-syn
? Same as above?	(Ulusoy et al., 2015).		mice or nonhuman primates			α-syn transfer occurs more efficiently in healthy neurons than in neurodegenerating ones	α-syn release occur in dead neuron rather than active neuron
2019 Vasali		a-syn fibril	WT mice	striatum	4 months	• 1m: (p-α-syn) aggregates in frontal cortex, SNpc, and the amygdala, primarily ipsilateral to the injection site (IHC, Figure 6B). • 6m: ↓ (35%) TH-positive cells in ipsi SNpc (IHC)
Atuka 예전		AAV-A53T aSYN		SN	4 months	↑ Ser129 aSYN in the caudate and putamen, ↓ DA neurons, ↓ striatal DA, ↓ DaT signal. However, there is no aSYN trans-synaptic spreading and no signal beyond the striatum
Atuka 예전		PFF + AAV-A53T aSYN	rodents			significant spreading
Atuka NHP		① [day 1] Bilateral injection in SN of AAV-hA53T aSyn → ② [day 28] Bilateral Injection of PFFs derived from mouse (for trans-synaptic spreading), (or monomer ie control in striatum, 1 month later	NHP (cynomolgus monkeys, approximately 9 years of age)			'success' : pSer129 aSYN signal beyond the striatum) with corresponding neuron loss. After 4m injection, all the monkeys were killed)

Atuka NHP — Results matrix

				Results
DA system	DAT	striatum	PET 18F-FPCIT [125I]-RTI-121's ARG	Reduced DaTScan signal
DA system	VMAT2	Striatum	PET 18F-AV133	2 occasions, baseline + week prior to necropsy
	DA, DOPAC, HVA	Striatum	HPLC
NDGN	TH cell	SN	stereology	reduction of TH+ DA neurons; no spreading pathology
	atrophy	Whole brain	3T MRI
		WM	DTI
a-syn	pSer129 aSyn	forebrain, midbrain, brainstem	qualitative histology
	Total aSYN (of primary interest for this study), aSYN oligomers, aSYN seed assays	CSF
others	t-Tau, p-Tau, Inflammatory BM

Uncertain Spans

location	transcription	uncertainty
NHP Results header	`Reduced DaTScan signal` cell only partially visible in the available crops	reads as written; the rightmost columns of the Results matrix are partly clipped at the right edge.