Microglia mono/co-culture	NC				TR06673219 potently inhibited nigericin-induced IL-1β and IL-18
Cell line	Primary	FD	Human MG are characterized by immunofluorescence with antibodies to CD11b (Mac-1), CD68, CD86 (B7-2), HLA-ABC, HLA-DR, and KCa3.1 such Some have been characterized by characterizing morphology and other characteristics microglia phenotype MJF WORKSHOP 202108: human microglia appear to be relatively good at recapitulating in vivo phenotypes for NLRP3 research	Neither side		TR06673219 potently inhibited nigericin-induced (IC50 = 4 nM) of PRC release and IL-1β secretion of human PBMC. There was no cytotoxicity in the assay condition. all compounds inhibited IL-1β in a dose-dependent manner; other candidate compounds (T9890, TR098 and other 2 compounds) will be evaluated.
	IPS	(normal)	Human iPSC-derived microglia (IPS microglia)	[유일하게 가능한 이상한 하이패스] 가능 활성화에 (단지 NLRP3 activation이 release을 induce해 well 도 검토함) PFF에 대한 reaction이 있을 수 있어, 유일하게 가능한 분에 대한 이상한 하이패스를 구상함. T_Wood Hospital이 활성화에 의한 알 수 있는 차원에서, the iPS activated by ATP (but not by LPS) (asyn PFF는?)		TR06673219 potently inhibited nigericin-induced IL-1β (IC50 0.8 nM and IC50 0.55 nM) The treatment in response to MJF NLRP3 inflammasome activation by human-iPS Syn PFF ASC Spec assay & Cytokine release (Phenovista/Talisman)
	Direct conversion	human monocyte-derived microglia-like cell, MDMG-like cells, MDMG, MDMS [Talisman?]	(human) MDMA derived microglia phenotype (Talisman?) (normal) microglia (cortical?) neuron co-culture	Alpha-syn PFF assay (with NLRP3 activation; please note we will need to investigation of time and effort. Trent/Wood) Talisman set: T-Talisman and CRL		(human iPSC-derived co-culture) TR06673219 potently inhibited nigericin-induced (IC50 1.27nM) and IL-1β (IC50 1.25nM) well are currently assessing the IL-1β/A-syn 18-month treatment in response to NLRP3 inflammasome activation by human a-Syn PFF

General order of priority

a-syn (mostly used) of TR06673219 in primary human microglia ~100 nM (IL-1b and IL-18), while that of primary mouse microglia is 11.3 nM. Team will use the appropriate microglia assays to adjust for species potency differences. Our NLRP3 inhibitors (NLRP3i) are inhibiting the function of NLRP3. The activation of NLRP3 inflammasome of inflammatory response requires two steps: firstly LPS for upregulation of NLRP3 mRNA/protein and secondly nigericin or ATP or other stimulant to activate the NLRP3 pathway, that leads to IL-1β maturation. The above approach is intended to use NLRP3 inhibition (NLRP3i) of activated microglia. We have been given at the same time as the stimulant. However, across in vitro assays we always incubate the compounds for an appropriate period of time (~15 min) in order to give the compounds enough exposure prior to the addition of the stimulant. We employ the LPS-priming-only paradigm here because the stimulant in our compounds is necessary for hindrance addition of compounds in vitro assays is not advisable as 1) stimulation of the inflammasome leads to cytokine accumulation in the supernatant, within an hour, that we measure and such an assay would be very messy 2) the compounds’ potency and decrease dramatically and 3) stimulants like nigericin (used in our assay) are cytotoxic and we need our compounds to inhibit the inflammasome function at the prime of the cells.

Imaging neuroinflammation

	TSPO	P2X7 receptor	CSF1R (colony-stimulating factor 1 receptor)	TREM	1 PET (Cipriani 2022 #2293)
definition	18-kDa translocator protein (TSPO), =peripheral benzodiazepine binding site (PBBS)) TSPO) is a highly conserved protein located in the outer mitochondrial membrane. expressed de novo during the activation of microglia [expression] (Betlazar, 2018 #2042) Constitutive TSPO: i) endothelilial cell 에 주로 많아 (microglia 에는 없고) 이것이 baseline signal 을 낼 것, ii) inflammation 시: activated microglia 에서 expression 한다는 보고 많음, 결국 signal-to-background window 가 문제임.	purinergic ion channel 7 receptor (P2X7R), a cationic, ATP-mediated ion channel	CSF1R, a microglia-specific marker, the expression of which is essentially restricted to microglia within brain. CSF1R is a cell surface protein in a subfamily of tyrosine kinase receptors activated by two homodimeric ligands; CSF1 and IL-34 [7]. CSF1R is the primary regulator of the survival, proliferation, differentiation, and function of microglia [8]. CSF1R directly controls the development, survival, and maintenance of microglia and plays a pivotal role in neuroinflammation [9-11]. The inhibition of CSF1R has been postulated as a way to treat a variety of inflammatory and neuroinflammatory disorders [14]. Regional differences in CSF1R expression and microglia phenotype have not been studied in detail, but expression analysis in mice has demonstrated enhanced levels of CSF1R in subcortical regions and lower levels in other regions of the brain [15]. It is yet to be clarified whether CSF1R is a marker of microglia in general (with a specific phenotype, but it is conceivable that both homeostatic and reactive microglial cells express CSF1R at variable levels.	[11C]CPPC, a CSF1R has been licensed to a Johns Hopkins start-up company, https://www.eccharlie.com/grants/development-stimulating-factor-1-receptor-csf1r-radioligand-screening (AKL to measure the ligand binding to human brain PET imaging.)
Ligand	(PET) (옛날유행!) (R)-[11C]PK11195, [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide : low signal-to-noise ratio, high nonspecific binding, low brain penetration, and high plasma protein binding The cellular location of the signal is another major concern for TSPO ligands. Two different binding sites on glial and vascular TSPO were reported for several TSPO ligands, e.g., [11C]PK11195 (57). Meta-analysis: (Zhang, 2022 #2287)	11C-JNJ-54173717 has undergone successful preclinical evaluation in rat and monkey brain and will undergo further evaluation in the clinic in neurodegenerative diseases (Ory et al., 2016).studies in NHP with 11C-JNJ-54173717 showed a substantial decrease in observed SUV upon self-block and blocking with chemically distinct P2X7 antagonist (JNJ-42253432) [109], 11C-JNJ-54173717 SMW139 have been characterised with a viral vector model which causes overexpressed human P2X7 in one hemisphere of rat, with control viral vector injected into the other. Both tracers showed increased uptake in the hemisphere with human P2X7 expressing viral vector and homogeneous binding with P2X7 [107, 109]. For 11C-JNJ-54173717 has been reported, and it demonstrated its utility in quantifying P2X7 expression in the CNS	(Mizumura, 2021 #2306) Automated reading of CPPC were prepared, and four were found to have high (sub nanomolar) potencies and selectivities to inhibit CSF1R. Neither radioligand showed defective specific binding to brain CSF1R as evidenced by self-blocking studies. 11C: [¹¹C]GW2580 [171], [¹¹C]CPPC (PMID 31171578), [¹¹C]CW3543 (PMID #1736) (mouse & NHP) GW2580 captured changes in CSF1R availability more sensitively than 11C-CPPC, with a larger dynamic range and a smaller intra-individual variability. I conclude that according to Dr Pomper's the GW2580 PET imaging may require more pre-optimization to be more forward to clinical development as if showed dose elimination in the brain (~30 min). Cell-free assay JNJ-42253432 11C-JNJ-54173717 11C-SMW139 (Pri., AON1 NJ) 2,4,4-c 4 (1.5%) (NC) self-blocking (in noncoated cells x 1.5(SD%) for both 11C-CPPC vs 11C-CPPC) 0 ng/ml -50 nM Are we allowed to do Radioligand study in PD postmortem human tissues? 외부 (대학 등) IRB pass 가능 (외부 thesaurus & selectively 사실) 안 됐던지 알 수 있을지 가짜 ID 가... Ashley Knight (LAG) is not yet IPDS studied [DIO], I performed a couple of ARG experiments using rodent and human tissue. I will be carrying out IHC on these tissues to characterize CSF1R expression. We are also planning to use human tissue from primary tumors. Based on what I do see so far, I'm seeing no better specific binding than [11C]CPPC in my own hands. Once I have these data in a summary format, I will share with the group.	In vitro studies Cell-free assay (FRET assay): Binding Affinity: IC50 (phosphorylation extent) of 3.42 nM (comparable to CPPC) 'well plate binding study' Binding confirmed in well plates coated with CSF1R (vs non-coated wells, x7 fold)
	2nd generation [18F]-FEPPA [3H]PBR28 , [11C]PBR28 11C-PBR28, 18F-PBR111, 18F-FEDAA1106, 18F-FEPPA, 18F-PBR06, 11C-DPA-713 and 18F-DPA-714 (N,N-diethyl-2-[4-(2-fluoroethoxy)phenyl]-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-acetamide , Meta-analysis: (Zhang, 2022 #2287) included fifteen TSPO PET studies in PD. The paper shows the summary results by generations of TSPO PET — 1st and 2nd. Please note that the analysis didn't include Yacoubian et al. 2023 (from Univ of Alabama, PMID 36853618). 18F-DPA-714 : modelling not to use arterial input: (García-Lorenzo, 2018 #2160) {Fang, 2022 #2161: Alabama group} (Boutin, 2013 #1764) Better signal-to-noise ratio (=target region/reference region) than PK11195 (Direct comparison in the same animal). Lavisse 2021 #2145 brain region heatmap panels: PD patients (n = 24, H&y I-III) and 28 HC, [18F]-DPA714 and [11C]-PE2I, No reference region, but no arterial input Supervised cluster analysis (SVCA), Results: PD patients showed significantly higher [18F]-DPA714 binding compared to healthy controls bilaterally in the midbrain (p < 0.001), the frontal cortex (p = 0.001), and the putamen contralateral to the more clinically affected hemibody (p = 0.038). Microglial activation in these regions did not correlate with the severity of motor symptoms, disease duration nor putaminal [11C]-PE2I uptake. However, there was a trend toward a correlation between cortical TSPO binding and disease duration (Yacoubian et al. 2022, https://doi.org/10.1101/2022.09.21.22280194) De novo PD patients vs HC: 184 De novo PD patients vs HC.
	(Rodríguez-Chinchilla, 2020 #2163) RAAV-BASED ASYN RAT MODEL (intranigral injection) ... predominant !. Correlation between DPA-714 and IBA1 cells: r=0.41 (significant), Correlation between DPA-714 and TH cells: r=0.45 (significant), Correlation between DPA-714 and GFAP cells: only r=0.17 (non-significant).

Uncertain Spans

location	transcription	uncertainty
Imaging neuroinflammation / TSPO definition / (Betlazar, 2018 #2042)	reads Betlazar; preserved verbatim though canonical author surname may be Betlehem or similar.	low confidence on author name.
CSF1R / (Mizumura, 2021 #2306)	reads Mizumura; preserved verbatim.	low confidence on author name.
CSF1R / radioligand list	reads [¹¹C]GW2580 [171], [¹¹C]CPPC (PMID 31171578), [¹¹C]CW3543 (PMID #1736); preserved verbatim.	low confidence on PMID alignment.