AGS/ALS/cGAS to Parkin/PRKN Transition

AGS/cGAS Evidence Table Continuation

The visible top rows are the lower part of a larger AGS/cGAS table. The partial column/header text includes:

accumulation of cytosolic self-DNA (exonuclease TREX1)
c self-DNA (mito DNA아님)
(cGAMP) 생성함
STING
IRF3
response genes in the nucleus
T-Cell Activation

block	model / evidence	visible note
Evidence / Human	no visible entry in this crop	Empty row or content above the crop.
Evidence / Mice	{Gao, 2015 #772} TREX1-deficient mice	In heart tissue, kidney and PBMC, sera: cytosolic accumulation of self-DNA (이건 논문에 없는 느낌?) and cGAMP (in heart tissue, using MS); ↑ mRNAs of ISGs (IFN-stimulated genes, by RT-PCR (qRT-PCR)), including IFIT3, CXCL10 (=IP-10), and IRF7, as well as the inflammatory cytokines TNF-a and IL-12.
Correction / mice	{Gao, 2015 #772} genetic ablation of cGas in Trex1-/- mice	Eliminated all detectable pathological and molecular phenotypes, including ISG induction, autoantibody production, aberrant T-cell activation, and lethality. Even deletion of just one allele of cGas largely rescued the Trex1-/- phenotypes of mice.
primary culture	{Vincent, 2017 #773} bone marrow-derived macrophages (BMDMs) from Trex1-/- mice	RU.521 reduces IFNb1, IL-6 mRNA, in the presence of immune stimulators: dsDNA, 5'ppp-HP20 RNA, Pam3CSK4, poly(I:C), LPS, or recombinant murine interferon-beta.
In vitro	{Vincent, 2017 #773} RAW cells	RU.521 reduces IFNB-1 (type I IFN) in RAW cells.

ALS with cGAS

Rationale

• (Neta's email 20200201) elevated levels of the specific cGAS signalling metabolite cGAMP in iPSC-derived motor neurons and post-mortem spinal cord samples of patients with ALS.

Mechanism

• (Neta's email 20200201) accumulation of wild-type TDP-43 and further exacerbated by mutant TDP-43.
• Mislocalized TDP-43 invades mitochondria via TIM22 and causes the mitochondrial Permeability Transition Pore (mPTP) to release mitochondrial DNA (mtDNA) into the cytoplasm.
• mtDNA directly activates cGAS → ↑ cGAMP → ↑ STING → upregulation of NF-kB and type I IFN → neuron loss.

Progress

• MNI is testing (neuronal cell death assay) our compound in 3D coculture of motor neuron (ALS patient-derived) with astrocyte.
• Yan; Neuronal cell death assay with ALS iPSC NM in EONU which is the same with MAP4K4/ferroptosis dual inhibitor program
• Will get results by Mar 2021

HD with cGAS

• Will be challenging

Parkin / PARK2 Gene (=PRKN)

Location

• Chromosome 6 in human
• Chromosome 17 in mouse
• Codes for the ubiquitin E3 ligase that is involved in …
• PRKN gene is located at 6q26, consists of 12 exons

Position

• Cytogenetic location: 6q26
• Genomic coordinates (GRCh38): 6:161,347,416-162,727,801 (from NCBI)
• Human PRKN has 8 mRNA transcript variants
• Variant 1, a major transcript, CDS used in program, 1398 bp in size, and encodes Parkin protein isoform 1

Expression

• Parkin is ubiquitously expressed
• Parkin is highly expressed throughout the brain including SN: https://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKN
• Parkin is the most abundant in neurons in the human brain (Jacobo, Sarah Melissa)
• Expressed in heart, testis and skeletal muscle
• Found in serum: https://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKN

PRKN Gene, Homology, and Expression Figure

204bp Shared Promoter
PACRG
Parkin
~2.0 Mbp
DOI: 10.1046/j.1471-4159.2001.00512.x
 
Human
Rhesus 99% identity with human
Mouse 94% identity with human
 
FPKM
Fetal Astrocytes
Mature Astrocytes
Neurons
Oligodendrocytes
Microglia/Macrophage
Endothelial

Splicing

• Human PRKN has 26 splice variants: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4124806/
• Variant 1 is the major transcript with 1398 bp CDS
• Variant 1 coding sequence (CDS) is used in this program
• Variant 1 encodes for Parkin protein isoform 1

Parkin Protein / Concentration of Parkin Protein

The table is partially cut at the left and bottom. Visible column groups:

Human: Brain / CSF / serum
NHP: Brain / CSF / serum

source / method	Human brain	Human CSF	Human serum	NHP brain	NHP CSF	NHP serum
Takeshi from the commercially available human brain / ELISA	771 (500-1500) pg/mg total protein	blank visible	blank visible	blank visible	blank visible	blank visible
TM: SMCxPro-/well; capture antibody: antiParkin abcam, rabbit mono; EPR18567 176 12.5 ug/MP mg, 5 ugMP/well; detection antibody: antiParkin abcam, rabbit mono; EPR18567 214 125 ng/mL	SMCxpro; (n=5) 1.3 to 22.7 pg/mg total protein; Mean: 6.92 pg/mg (=7ng/g=0.007ug/g) 적네!; SD: 8.894; Warning: sample size is less than 30, you should be using the POPULATION standard deviation.; 80% CI: 6.92 +/- 5.1 (1.82 to 12); "With 80% confidence the population mean is between 1.82 and 12, based on only 5 samples."; Cf) {Quinn, 2012 #2128} aSyn (LC-MS): 500 ug/g protein	SMCxpro; n 7; Minimum 0.7000; Maximum 2.500; Range 1.800; Mean 1.257; SD 0.5740; SEM 0.2170	blank visible	Monkey brain sample will be evaluated using current parkin protein assay method in August to confirm whether our assay method detects monkey parkin protein or not.	SMCxpro	No signal
MS by DMPK / LC/MS	3590 - 9052 pg/mg protein	bottom continues below visible crop	bottom continues below visible crop	bottom continues below visible crop	bottom continues below visible crop	bottom continues below visible crop

Uncertain Spans

location	text/status	reason
AGS/cGAS table top	partial mechanism columns	The table begins above the visible crop; only lower continuation can be reconstructed here.
Parkin location	codes for the ubiquitin E3 ligase that is involved in ...	Sentence is cut off after involved in; no completion inferred.
antibody names	EPR18567 176, 12.5 ug/MP mg, EPR18567 214, 5 ugMP/well	Small assay-method text is high-risk; spacing and unit formatting were updated from the raw photo but should be checked before assay reuse.