pS65-Ub Postmortem Evidence, NBB Comparison, And Assay Development Notes

Hou 2018 p-S65-Ub Aging And LBD Figures

Source/context note:

(Hou, 2018 #819) Parkin PD postmortem brain (n=7, SN), PINK2-PD (n=2)
Mayo Clinic Florida Brain Bank, IHC
 
In vitro: ↑ functional parkin
(method: HeLa cell overexpressing WT parkin ie 'HeLa Parkin')
in HeLa cell (in the presence of CCCP)
 
postmortem sPD, correlating with Braak stage & age
(나이 들수록 damaged mt 많아지지만, mitophagy는 functional 하니까 이겠지)
 
advanced PD > early PD > old normal > mid-aged normal >
(young normal = Parkin-PD = PINK1-PD)

Figure labels:

Figure S4
Neurologically normal aging
LBD
SN p-S65-Ub cell density
Hippocampus p-S65-Ub cell density
Amygdala p-S65-Ub cell density
Putamen p-S65-Ub cell density
nbM p-S65-Ub cell density
Age
Braak tangle stage
Thal amyloid phase

Caption fragment near the upper-right figure:

Figure 4. p-S65-Ub cell density increases with advanced aging and Braak
tangle stage in select brain regions in neurologically normal controls or
in LBD.

Summary Table

Marker / question	Parkin PD	normal control	sPD
Ps65ub SN	0	0.2 (no age-depedency)	0.7 (no age-depedency)
Ps65ub other area (eg putamen)		age-depedency)(50 세 putamen 에서 거의 0, fig S4)	age-depedency)(50 세 putamen 에서 거의 0, fig S4)
CSF?		Js: Might be age-dependant due to the contribution of large regions (eg. putamen), so there is a need to confirm difference between groups in the CSF of middle aged subjects.

Hou 2018 Reduced Or Absent p-S65-Ub Staining Figure

Caption text:

Figure 2. Reduced or absent p-S65-Ub staining in brains from PRKN or PINK1
mutation carriers.

Figure/caption anchors:

control
sporadic
PRKN
PINK1
PD
p-S65-Ub cell density
 
SN sections from controls, sporadic LBD and cases with PINK1 or PRKN
mutations were stained with p-S65-Ub antibody.
 
Representative images of stained sections show enhanced p-S65-Ub levels in
sporadic PD, but reduced or absent signal in SN of PRKN or PINK1 mutant cases.
 
(B) Quantification of p-S65-Ub signal in age-matched controls, sporadic LBD
cases and mutation carriers.

Note beneath the figure:

(Hou, 2018 #819) this figure is SN, control means 'age-matched controls',
n=17, mean age 68 y

Shiba-Fukushima 2017 Phospho-Ub IHC Note

Source/context note:

참고: (Shiba-Fukushima, 2017 #1057) Postmortem, SN
phosphoUb 를 봤으나 ps65-ub 라고는 안말함.
phosphoUb: control (young & old)에서 없음, Park2 & Pak6 에 없음.
MSA 에 약간 있음.

Image labels:

Control
PARK2
sPD
Control (young)
PARK2
PD
TH / phospho Ub / poly Ub

Expected Treatment Effect And PRKN-PD Scenario Notes

예상 Tx effect in young HC and young NHP:
barely detectible -> still barely detectible
(damaged mitochondria 적으므로)
 
In PRKN-PD: barely detectible -> scenario
i) if mitophagy 의 정상적범위에서 처리가능하다면 no increase,
ii) if mitophagy 급증 혹은 여전히 mitochondria are damaged, then increase

NBB Sample Brain / CSF Values

NBB sample	CSF	Brain
PARK2	PARK2-1: 2.3 PARK2-2: 3.3	PARK2-1: 7.3 PARK2-2: 4.4
sPD (old age 명심!)	6.4±0.9*	11.14±3.01*
HC (old age 명심!)	6.4±3.4*	16.78±8.06*

NBB Brain / CSF pS65Ub K48 Plots

Plot labels:

NBB Brain
pS65Ub, K48 (pg/mg total protein)
PARK2
sPD
HC
 
NBB CSF
pS65Ub, K48 (pg/mL)
PARK2
sPD
HC

Interpretation Notes Before Next-Step Plan

정상적인 상황에서 없고, mitophagy 활발히 필요하나
PINK1 없으면 없음.
 
In vitro: mitophagy 활발히 필요할 때 증가 (eg. aging)
In vivo: detected when incomplete degradation
 
In Young 과 Old sporadic PD?: 에서의 DATA 없는 듯
(young 이니까 없으려나? PD 에서 mito damage 많지만 mitophagy 는 잘되고 있다면 ps65ub 많을 것, PINK1-)
 
PARKIN pathway 가 원인측에 있다면 ps65ub 적을 것?
 
Parkin-PD?: PARKIN 적으니 이것도 적으려나?
2018 Ordureau fig 6, in vitro) 토대하면 적겠다.
 
어쨌든, mito damage (by CCCP) & (exog) Parin 주면 -> ↑

Next-Step Development Plan

Area	Plan text
Detection assay platform	Detection assay platform
Disease relevance	위를 이걸 꼭 몰라도, ie disease-relevance 모르더라도, (tubulin acetylation 경우처럼) proximal PD marker 로 사용하면 될 것 같으니, post-mortem brain 검사 안 해도 될 듯하고, 이건 안 중요하지 않나?
(Proximal) pathway relevance	Correlation with mitophagy?, 중요함. 그런데 증명해야 하나? - In vitro: Fiesel 은 ps65ub 만 보고, 2010 Ding 은 mitophagy 만 봤으니, 둘을 한꺼번에 본 것이 없는 듯 한데? 우리는 iPS 에서 (아래 treatment relevance 와 함께) 해보자. - In vivo: AAV-PARKIN 으로 보여야 하겠지.
Treatment relevance	Level change in response to WT exogenous parkin - In vitro: (cell line 이면 transfection 시키면 되는데, Ips 는 어떻게 exogenous parkin 을 존재시키나? Recombinant parkin 은 cell 진입이 어려우므로 못 쓸테고, AAV 쓰면 될 것임. - In vivo: AAV-PARKIN 으로 보여야 하겠지

Correction Of pS65-Ub / External Assay Notes

Correction Of pS65-Ub

Readout / model	Source	No CCCP, No parqin	CCCP only	Exogenous Parkin only	CCCP + Exogenous Parkin
pS65-Ub; HeLa cell	(Fiesel, 2015 #700) fig2a	Not detectible	↑	Not detectible	↑↑
mitophagy; HeLa cell	2010 Ding	A little	↑ (fig 1)	No increase	↑
HCT116 cells	2010 Ding		↑
MEF cells	2010 Ding		↑
Fibroblast from Parkin-PD	(Koentjoro, 2017 #711)		No change 인 듯 (fig 4c)
Fibroblast from Parkin homoz mutation but no PD	(Koentjoro, 2017 #711)		To be checked!

External Assay Notes

Ub assays (external)
Takeda
 
Capture Ab
Ubiquitin
mouse
mono, P4D1 (CST, 3936S),
 
Detection Ab
Phospho-ub
(K63 pUb tetra?)
rabbit poly
 
Standard protein -
K63 pUb tetra

Uncertain Spans

• PINK2-PD (n=2) in the Hou 2018 source note conflicts with nearby PINK1 figure labels.
• Hou 2018 figure panel labels, axis values, Spearman r, p-values, and sample counts are image-primary; use the embedded figures before extracting exact numeric values.
• NBB table values, plus/minus values, asterisks, group labels, and axis units are small and uncertain in the photo.
• Column/row alignment in the correction table is uncertain because the table is partially cropped at the lower edge and photographed at an angle.