Parkin Fibroblast Translation Plan And Parkin Delivery Evidence
Fibroblast, Lipid, And iPSC Evidence
General/fibroblast notes:
Matching Control feasible?
doable, but the same passage number, doubling time, culture condition matters will be challenging.
(Imaizumi, 2012 #1328) In PARK2 iPSC-derived neurons, but not PARK2 fibroblasts or iPSCs, abnormal mitochondrial morphology
(highly electron-dense matrix and swollen mitochondrial cristae within the inner mitochondrial membrane (IMM)) (Figure 3A, black arrowheads).
(Mortiboys, 2013 #1158) genotype available (Takanori slide), Ursocholanic acid tested.
(Lobasso, 2017 #1157) genotype available, n=2, some phospholipids and glycosphingolipids were altered in the lipid
profiles of parkin-mutant fibroblasts. The detected higher level of gangliosides, phosphatidylinositol, and phosphatidylserine
2008 Mortiboys: skin fibroblasts from parkin mutant patients may be a suitable system to test new therapeutic approaches, at least
those based on rescue of mitochondrial phenotypes,
- display signature alterations in mito
- the cell damage at the age of the patient (Zilocchi 2020)
- basic mechanisms active in neural cells in PD are likely expressed in fibroblast iniPSC row anchors:
| Field | Content |
|---|---|
| Model | iPSC |
| Mutation/genotype | c.1072Tdel; p.A324 fsX110; Homozygous |
| Chung et al. 2016 | Fig 2a/b/c; ↑ mito size; ↑ mitochondrial superoxide levels in ips-DA neurons from patients with mutations in either gene (PINK1, PARK2) |
| Shaltouki et al. 2015 | reduced mitochondria volume in ips-DA neurons with PARK2 mutations |
| Suzuki et al. 2017 | ↓ mitophagy (fig4d) |
| Jiang, 2012 #1356 | iPSC from Park2-PD patients; ↑ transcription of monoamine oxidases and oxidative stress; ↓ DA uptake & ↑ spontaneous DA release; corrected by lentiviral expression of parkin |
| Okarmus, 2020 #1331 | isogenic iPS; proteomic analysis; 119 proteins altered; Swollen, mito with ↓ matrix density and irregular cristae; increased TOM20 (Fig4b) |
| Bogetofte, 2019 #1332 | PARK2 KO did not significantly affect the total number of mitochondria as measured by TOM20 immunofluorescence staining (Figures 4A,B); significantly increased area of TOM20 immunoreactivity; overall larger mitochondrial area per cell (Figures 4A,C) |
| Kumar, 2020 #1333 | ↓ OCR; ↓ MMP; ↓ TOM20 (fig7n) |
| Rakovic, 2015 #1334 | review |
Comparison note at the left:
Cf) (Grünewald, 2016 #935) sPD (parkin-PD 아님!) postmortem brain,
Whereas mitochondrial mass was unchanged in single SN neurons from IPD patients,
we observed a significant reduction in the abundances of CI and III? subunits in dopaminergic neurons
- fewer transcription/replication-associated mtDNA molecules
and an overall reduction in mtDNA copy number
Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α): master regulator of mitochondrial functions and oxidative metabolismTranslation Plan Fibroblast And Leukocyte Complex Activity
Translation Plan Fibroblast
| Question | Stage / requirement | Evidence note |
|---|---|---|
Fibroblasts from Parkin-PD show ↓ mitophagy? | 대표적 흔한것들에 필요 | Papers exist (Koentjoro, 2017 #711) |
If yes, Corrected by exogenous Parkin? | Before clinical trial; 대표적 흔한것들에 필요 | No evidence but T. Nishimura in NS-DDU PhRET showed this in PARK2 KO iPSC-DA neurons (is this patient-derived?) |
Clinical trial | row label |
Leukocyte
Leukocyte
(Müftüoglu, 2004 #1027) 10 patients from 6 families with parkin gene mutations,
20 patients with idiopathic PD, ...
Method: venous blood samples collected -> WBC isolated -> mito isolated (fractionation) 즉 culture 아님!
Results: ↓ MC I and IV activities in leukocytes from patients with idiopathic PD,
and decreased complex I activity, with normality of complex IV, in patients with parkin
mutations (by 62.5%) and Spd (by 64.5%): table 2, fig1 (seems clearly differentiated)Correction By Parkin In Vitro
| Study / row | Cell/model | Intervention / note | Readout notes |
|---|---|---|---|
(Ding et al. 2010, PMID 20573959) | HeLa cells (endogenous parkin level was very low, not neuronal, js: cervical cancer origin이니 muscle cell 인가?) | Exogenous parkin (transfection of cells with parkin), AAV 등을 써야지, 그냥 (recombinant) protein 주입은 안 될 것임 (cell 내로 안 들어가므로) | Fig 2a & b; ↑ mitophagy (in the presence of CCCP, mtKeima미사용); ↑ mito ubiquitination -> ↑ p62 recruitment to mito -> ↑ co-recruiting the p62-associated LC3; ↓ the number of mito (ie ↑ Tom20, a mitochondrial outer membrane molecule); ↓ the size of mito (?, how to confirm this?); ↑ co-localization of GFPLC3 puncta with the mitochondria; recognition of the mitochondria (ie 'mito priming'); No effect on the total number of GFP-LC3 ... |
(Narendra et al. 2008, PMID) | HEK293 cells + CCCP (10 μM CCCP for 1 h) | mostly blank | redistribution of YFP-Parkin from the cytosol to the mito; Fig5; ↑ mitophagy ... LC3-positive autophagosomes in >30 cells per condition in at least three independent experiments; mtKeima미사용 |
T. Nishimura in NS-DDU PhRET | PARK2 KO iPSC-DA neurons (is this patient-derived?) | mostly blank | Parkin mRNA overexpression enhanced mitophagy (ie co-localization of GFP-LC3 and RFP-Mito); RFP-Mito labels Pyruvate dehydrogenase (lipoamide) alpha 1 ... |
(Koentjoro et al. 2017, PMID) | Fibroblast from PARKIN-PD | ↑ NIX (by pharmaco-logical induction, or Transduction of Nix-FLAG lentivirus in fibroblas[t]) | NIX -> ↓ mito mass, ↓ mito DNA (dose dependency는 안 봄, under CCCP, ie ↑ mitophagy, Fig 5B,C, fig 4d,e) & colocalization with LC3 (Fig4c) |
Parkin GT | Fibroblast from PARKIN-PD | Exogenous parkin | Mitophagy 이걸 본 것은 없네? |
Parkin Gene/Protein Delivery Evidence In Animal Models
Review rows:
(2019 Bulck) In a variety of preclinical studies in PD animal models, it has been described that gene therapy consisting of the overexpression of PINK1 and parkin proteins has neuroprotective effects by restoring or enhancing mitochondrial function and bioenergetics.
(Yasuda et al. 2011, PMID 21760537) 2nd, ectopically overexpressed parkin provides neuroprotective effects in genetic and environmental
PD models, including LRRK2-transgenic (23) and PINK1-knockdown fruit flies (24), 6-hydroxydopamine-lesioned rats (25), and ...| Model | Study | Intervention / timing | Outcome notes |
|---|---|---|---|
MPTP mice | (Paterna et al. 2007, PMID 17299411) | mice treated transiently with MPTP; (preventive, Parkin overexpression데); AAV2-Parkin into unilateral SN; after 21-26 days; MPTP Injection (ip, 4 consecutive); 7 days after MPTP, killing -> analysis | prevented DA cell loss; But no effect on striatal DA, DOPAC, HVA (fig 2b,c,d); surviving TH neurons may be compromised in their ability to synthesize dopamine |
MPTP mice | (Yasuda et al. 2011, PMID 21760537) | chronic MPTP-Minipump mouse; (preventive, Parkin overexpression데); AAV-Parkin into unilateral SN; after 14 days; MPTP pump implantation; At 7 or 28 days after implantation of the minipumps, killing -> analysis | prevented motor deficits & DA cell loss; But no effect on striatal DA, DOPAC, HVA (fig 4a,b,c); possible discrepancy from enhanced dopamine release of the surviving DA neurons that overexpress parkin |
6OHDA Rat | (Vercammen et al. 2006, PMID 16914382) | Lentiviral vectors encoding human WT parkin were stereotactically injected into the SN 2 weeks prior to a striatal 6-OHDA lesion; preventive | no report on DA, DOPAC, HVA |
6OHDA Mice | (Chung et al. 2020, PMID 32494688) Cellivery | iCP-Parkin (iCP=intracellular delivery of CPP-Parkin protein); (iv delivery) in 6OHDA mice/Rat or AAV-aSyn mice model (pff 용어는 미사용) | Korean note below |
Chung/Cellivery note:
Takeshi) To overcome solubility issue (otherwise precipitate) -> they added solubilization domain
to produce iCP-Parkin. Even though they adopt this strategy, they couldn't produce soluble
protein in E.coli. Although they refold insoluble iCP-Parkin from the inclusion body, iCP-Parkin
has not a correct structure, resulting in constitutive active protein (?).Korean interpretive note on iCP-Parkin row:
Parkin-KO Mouse 아니니 parkin level 안 봄!
1. Mouse: (fig5A), 6OHDA unilateral lesioning -> 4일 후 ICP-PARKIN (for 3 days)
(icp-parkin 직전에) apomorphine rotation test로 확인했으니 (FIG5E, DA loss 도 봤고)
Symptomatic 이네 (이 시점에 ↓ DA일것) therapeutic! -> 6ohda lesioning으로부터 12일 후 kill
-> TH level을 아예 No Tx group의 90%수준으로 만들었네,
(figS6d) restored COX4 & VDAC1, pole test에서 slowing progression 아니라 치료첫날부터 호전시켰고,
시간이 지나면서 vehicle 군은 거의 불변인데, 치료군은 BL보다 improved. (fig5c)
2. Rat: (fig5G), 6OHDA unilateral lesioning -> 2w 후 ICP-PARKIN (for 4 w)
(icp-parkin 직전에) apomorphine rotation test로 확인했으니 (FIG5j,k, DA loss 도 봤고)
Symptomatic 이네 therapeutic! (이 시점에 ↓ DA일것) -> 7주후 kill -> TH level을
No Tx group의 80-90%수준으로 만들었네 normalized DA loss, rotarod 에서 slowing ...Uncertain Spans
nav_path: inferred asParkin > Parkin PD, but body rows includeParkin GT.Grünewald, 2016 #935:CI and III? subunitsis uncertain; the second complex may be another Roman numeral.c.1072Tdel,p.A324 fsX110, and mutation/genotype labels in the iPSC row are small and uncertain.Müftüoglu, 2004 #1027: patient counts,62.5%,64.5%, and whetherSpdmeans sporadic PD are uncertain.MC I,MC1,complex I, andComplex IVterminology is preserved close to the source text.- Ding/Narendra/T. Nishimura rows: figure labels, mtKeima use/non-use, GFP-LC3/p62/RFP-Mito labels, and arrow directions are small and uncertain.
- Koentjoro/NIX row:
↓ mito mass,↓ mito DNA, dose-dependency note,Fig 5B,C,fig 4d,e, andFig4care uncertain. - In-vivo rows: PMIDs, MPTP/6-OHDA model names, AAV/lentiviral/iCP routes, SN/striatal lesion timing, injection/killing timelines, and DA/TH/DOPAC/HVA outcomes are small and uncertain.
- Korean notes in the Chung/Cellivery row contain OCR-sensitive phrases such as
불변,치료군,BL보다 improved, and bottom-truncated rat outcome text.