a-syn POSTMORTEM, a-syn SH-SY5Y + Cullen, a-syn PC only, FRACTIONATION

Choi 2011 요약

의미: 1. Monomeric a-syn doesn’t change in PD or GD or PD+GD or PD with GBA hetero

source	fraction	readout	normal	synopathy only	(GBA mutation homo or hetero) + synopathy	GBA mutation homo only
Choi (cerebral cx homogenates) 2011	TBS soluble (soluble, cytosolic)	monomer			Normal과 같음.	Normal과 같음.
	TBS soluble (soluble, cytosolic)	Oligomer	약간	Normal과 같음	Normal과 같음.	Normal과 같음.
	SDS soluble (soluble, memb bound)	Monomer		normal보다 증가	normal보다 증가	normal보다 증가
	SDS soluble (soluble, memb bound)	oligomer	absent	present	Present	absent (의미: neither GBA deficiency nor mutation alone result in a-syn aggregation)
	Urea Soluble (insoluble)	Monomer			Normal과 비슷함	Normal과 비슷함
	Urea Soluble (insoluble)	oligomer	없음	normal보다 증가	normal보다 증가	없음 (의미: neither GBA deficiency nor mutation alone result in a-syn aggregation)

Postmortem rows (Mazzulli 2011, Murphy 2013, Murphy 2014)

source	fraction	readout	normal	synopathy only	(GBA mutation homo or hetero) + synopathy	GBA mutation homo only
Mazzulli (bidirectional) 2011 Postmortem	TBS soluble (soluble, cytosolic)	monomer		이 군은 없었음.	Homoz: nl보다 ↑ Hetero: nl과 같음	Normal과 별 차이 없는 듯 보임.
Mazzulli (bidirectional) 2011 Postmortem	TBS soluble (soluble, cytosolic)	Oligomer		이 군은 없었음.	Homoz: nl보다 ↑ Hetero: nl보다 쬐끔(?) ↑	Normal과 차이없음. (absence of oligomer 라고 본문에서 시인했음.)
Murphy 2013 Postmortem	TBS soluble	monomer	TBS soluble data not shown, but only mentione that SDS soluble fraction increased compared to TBS soluble, indicating A-syn become insoluble (the shift from soluble to more insoluble) 그러나, SDS soluble도 여전히 soluble 하므로, solubility 차이보다는 localisation차이로 설명하는 게 낫지 않나?		Not applicable
	TBS soluble	Oligomer	Not shown		Not applicable
	SDS soluble	Monomer		↑ than normal	Not shown
	SDS soluble	oligomer	Not shown
Murphy 2014 Postmortem	TBS soluble	monomer		No change (synopathy only, ant cingulate cx & parahippocampal cx)	Not applicable
	TBS soluble	Oligomer	Not shown		Not applicable
	SDS soluble	Monomer		↑ than normal (synopathy only, ant cingulate cx)	Not applicable
	SDS soluble	oligomer	Not shown		Not applicable

Zhou : Postmortem, PD brain에서 insolubility 증가함. (SDS soluble 과 urea soluble 다 같이 얘기하는 것 같음)

a-syn SH-SY5Y + Cullen: probably this is all for cell line]

row / model	Cleeter	Manning-Bog (2009)	Dermentzaki (2013)	Cullen
duration	30 days	2 days	7 days (unfractioned) 7 days
concentration	50uM	50-200	50,100,200uM 200uM	10,50,100uM (<1h or 24h)
SH-SY5Y indiff / monomer	↑ (unfractioned)	No change (unfractioned)	-No change (unfractioned, collection시 TX썼으니 membrane-bound도 모았을것임) Not done	Soluble fraction (ie cytosolic + memb) 이건 PC12에서만 CBE 썼고, ELISA로 total concentration 봤음. (ie both monomer & oligomer)
SH-SY5Y indiff / oligomer	Not done	NA	안 보여줌	Results: mutant forms increased SNCA levels (not all인데?) but the wild-type (ie CBE) had variable effects
SH-SY5Y diff / monomer	Not done	↑ (unfractioned)	No change (unfractioned, collection시 TX썼으니 membrane-bound도 모았을것임) No change (both cytosolic & memb bound)	GBA activity 낮은 경우들은 WT+CBE 밖에 없네. 여기쓰인 GBA mutant 들은 다 GBA Activity가 안 낮으니, overall 참고자료에서 빼는 게 안 낫나?
SH-SY5Y diff / oligomer	Not done	NA	안 보여줌 No change (both cytosolic & memb bound)

a-syn PC only

source / row	note
Mazzulli (2011)	CBE NOT USED (ie mouse primary cortical neuronal cultures, in which GBA had been partly depleted via RNAi, (보통 KD라고 하네?) and human iPS neurons derived from a GBA mutation carrier)
same / 1.	mouse primary cortical neuronal cultures, in which GBA had been partly depleted via RNAi (resulting in 50% ↓ GBA expression):
same / 1.a	↑ (1.8 fold) monomer a-syn (fraction not specified), (oligomer not shown),
same / 1.b	Tx-soluble (fig 3D): ↑ monomer, ↑ oligomer (by native SEC/WB)
same / 1.c	Tx-Insoluble (fig 3E): ↑ oligomer, (monomer은 별차이 안 느껴짐)
same / 2.	iPS cells derived from skin fibroblasts of a GD patient (containing <10% dopaminergic cells (fig 2E): ↑ (dramatic increase) monomer a-syn (TX-soluble, unfractioned), (oligomer not shown)
GBA activity assay	not required
Dermentzaki (2013)	Rat cortical neuron cultures (E18)
same	아래 넷 모두 control과 no difference : 모두 monomer, soluble, unfractioned lysates (collection시 TX썼으니 membrane-bound도 모았을것임)
same	-100uM 7d / -100uM 10d / -200uM 7d / -200uM 10d
Osellame	알고 봤더니 in vivo (midbrain)

FRACTIONATION

fraction / row	protocol	notes
won normal	50mM Tris-HCl, 1% SDS / Boil / Spin: 13,000g for 10 min / supernatant	boil하고 oligomer없겠구만. / 약짧spin이니 / 이건 unfractionated sample 이라 할 수 있겠는데, (ultracentrifugation 안 했으나) 1% SDS 썼으니 protein 이, membrane에서 remove 되어 녹아 있는 상태이니, cytosolic과 membrane form 이 같이있겠구만.
TBS-soluble	TBS buffer / Spin: 1,000g for 4 min→Sup / Spin: 100,000g for 1h→sup	Murphy2013: the same and labelled TBS-soluble as well. / Murphy2013: this contains cytosolic proteins or soluble / Spin: 1,000g for 4 min 통해 unbroken cell등 제거, Spin: 100,000g for 1h to sediment membrane-bound organelles / 내거: 여기서 monomer도 나왔음.
SDS-soluble	위 pellet → TBS, 5% SDS, 1% Triton X-100 → Spin: 100,000g for 30min→sup	1. Murphy2013: similar except no T-X100 and labelled SDS-soluble as well. / 2. Dermentzaki: similar except no SDS and labelled TritonX-soluble as well / Murphy2013: this contains membrane-associated proteins
SDS note	(WIKI: SDS) can be used to keep membrane proteins in solution during purification; however, because SDS causes denaturation, milder detergents such as Triton X-100 or CHAPS can be used to retain the protein's native conformation during complete purification.
한 것 / urea-soluble	위 pellet → 1X TBS w/ 8% SDS & 8M urea (by sonication)	Murphy2013: 'detergent insoluble urea-soluble fraction' / Choi: the same and "urea soluble" / Murphy2013: this contains resolubilized protein aggregates or 'insoluble' proteins. / Urea strongly denatures proteins.

Bottom continuation

source	visible cells
Gegg 2015	in areas of the brain with low asyn pathology 라고 언급있을 뿐 그 level은 언급 없음.
Gegg 2012	↓ gba activity & expression(?) in PD; adjacent cell: a-syn or mitochondrial dysfunction → ↓ GBA activity
Parkkinen 2011	PD+heterozygous GBA mutation VS PD only; adjacent cell: No difference in the number of LB aggregates; note: Normal 과 비교한 게 아니니까 당연한 결과 아닌가?
Murphy

<table Below by Sidransky 2012 review>

Author	Model	Result	Criticism
(2013)	(undifferentiated) 1. SH-SY5Y + CBE	1. ↓ Mito functio

Uncertain Spans

location	transcription	uncertainty
Choi 2011 / SDS soluble oligomer	`absent` / `present` / `Present` / `absent` placements	The cells are small with red `absent`/`Present` text; row/column placement was inferred from visual alignment.
FRACTIONATION / leftmost labels	`won normal`, `한 것`, `위 pellet`	The leftmost labels are clipped at the photo edge; partial Korean labels are preserved as visible.
FRACTIONATION / urea row	`8% SDS & 8M urea`	The original transcription showed `& &` (doubled `&`); the duplicate was likely an OCR artifact and is reduced to a single `&`.
Bottom continuation	`(2013)` row in the Sidransky table fragment	The first visible Sidransky row’s author column is reduced to a year `(2013)`; the full author/title is in `20240722_182146`.