mouse
Figure 1. Validation of the Nlrp3^L351P^/Tmem119^CRE^ mice. A-C. Nlrp3^-/-^ mice harboring Tmem119^CRE^ were bred with mice harboring the functionally null homozygous Nlrp3^L351P^ allele. D. Representative genotyping with PCR-based detection of the Tmem119^CRE^ allele. E. mRNA was extracted from the mesencephalon of otherwise Nlrp3^-/-^ mice treated with Tamoxifen for 7 days to induce the expression of Tmem119^CRE^, removing the stop codon, and activating the Nlrp3^L351P^ allele (see A-C). Data demonstrate complete loss of Nlrp3 expression in the Tmem119^CRE^ and Tmem119^CRE^/Nlrp3^L351P^ backgrounds (lanes 1 and 2). Data demonstrate tight tamoxifen (Tx) inducibility in the Tmem119^CRE^/Nlrp3^L351P^ backgrounds (lanes 3 and 4). H. Plasma G-CSF levels detected using ELISA in Tmem119^CRE^/Nlrp3^L351P^ mice. treated with LPS indicate tight control and functionality of the Nlrp3^L351P^ allele with no leakage or Tx-related background in control animals.
microglia-specific GOF mouse model (this is different from CINCA since it is only microglia), (unpublished, Tmem119-Cre, Nlrp3L351P -KI )
- MJFF fully executed the grant Takeda and Dr. Havrda coapplied
Tamoxifen-inducible Nlrp3 allele.
We developed a new mouse model by combining a recently developed microglia specific CRE-driver with a CRE-dependent hyperactive Nlrp3^L351PneoR^ allele based on mutations identified in patients with cryopyrin-associated periodic syndromes (CAPS) (Bonar et al. 2012, PMID 22558291, Katharine M von Herrmann 2020, PMID) (Fig. 1). In the Nlrp3^L351PneoR^ strain, the endogenous Nlrp3 gene is functionally null prior to CRE recombinase-mediated deletion of a reverse oriented neomycin cassette. We bred the Nlrp3^L351PneoR^ strain to homozygosity to eliminate the wild-type allele and crossed with microglia directed Tmem119^em1(cre/ERT2)Gfn^ mice (stock #031820) that we had backcrossed onto an Nlrp3^-/-^ background (Fig. 1). Treatment with tamoxifen results in disinhibition the hyperactive Nlrp3 allele, expressed only when the endogenous murine Nlrp3 gene would normally be induced, in these otherwise Nlrp3^-/-^ mice.
We have verified a robust neuroinflammatory response in tamoxifen treated Tmem119em1(cre/ERT2)Gfn/ Nlrp3L351PneoR mice (Fig. 2)
Figure 2. Tamoxifen inducible Nlrp3-dependent neuroinflammatory phenotype. Three-month old Tmem119^CRE^/Nlrp3^L351P^ mice activated with tamoxifen or vehicle alone were treated using 3 consecutive daily injections of 5mg/kg i.p. LPS. Tissues were harvested 72 hours after the final dose and histologic sections of the mesencephalon were immunostained for Iba1 and GFAP. Left panels were treated with vehicle, and are functionally Nlrp3^-/-^. Limited staining of ramified microglia (*upper left) and astroglia (lower left) are noted. Right panels were treated with tamoxifen to disinhibit the hyperactive Nlrp3^L351P^ allele (see Fig. 1E). In these section supernumerary, bushy* and amoeboid** microglia were observed (upper right) along with easily observable astrogliosis (lower right).
- using 3 consecutive daily injections of 5mg/kg i.p. LPS.
| Section | Notes |
|---|---|
| Original plan |
Plan: To test three compounds: - poor brain penetrance (MCC950) - better brain penetrance (Inzomelid) - in-house compounds READOUT? to develop a tractable biomarker strategy for related clinical trials Neuroinflammation will be analyzed post-mortem using immunohistochemistry. IBA1 staining will label microglia and GFAP will label astrocytes. Pro-inflammatory glial morphology will be determined as we report (Martinez et al. 2017, PMID 28903492, von Herrmann et al. 2020, PMID 32680528). Cellular specificity will be determined using co-immunofluorescence, labeling NLRP3, inflammasome activation related antigens like cleaved GSDMD and CASP1, and neuronal and glial markers. Extracts from a portion of matched hemispheres will be evaluated using bead-based cytokine multiplexing confirmed using ELISA and SDS-PAGE using SDS-PAGE to detect NLRP3, IL1B, and ASC and cleavage of CASP3, CASP1, IL1B (Fig. 3), and GSDMD. We will analyze inflammasome particles by blotting for NLRP3 and ASC in non-denaturing TBS conditions |
| Plan 20220528 Ex vivo |
Takeda will will send 2 compounds on the top of MCC-950 to test ex-vivo and on the mice. A) Test compounds ex-vivo on primary mouse microglia, +/- activation to demonstrate reduction of IL-1b levels — MCC-950 & — Takeda compounds (should be two) 20220528 Matt: Microglia culture protocols are working extremely well. Next litters of L351P are expected in about 14 days. That means ex vivo experiments in about 24 days. Should be plenty to try the 32dg. option. Will we have compounds by then? 20230112 Matt: low signal of IL-1b, moderate reduction by all three compounds below) |
| In vivo |
B) The compounds that show inhibition of the NLRP3 L351P mutation ex-vivo will be administered in vivo to examine • modulation of the NLRP3 pathway (NLRP3, ASC, Caspase-1, IL-1b/IL-18) in brain, plasma • modulation of other inflammation pathways (see below) C) Blood, Brain, Spleen, Liver tissue from the mice in (B) will be collected for analyses: |
| Methods |
— Gene Expression Panel: Nanostring: LBL-10402-V2 (Dr. Havrda's Lab) → 20220528 Matt: As far as adding to the Nanostring panel, we use XT_PGX_MmV2_Inflammation_CSO, Is LBL-10402-V2 a different panel? Keep in mind that we generate plenty of high quality RNA. We can run other panels, measure using QT-PCR, or do bulk RNASeq as needed. — Protein Expression Panel: Luminex Mouse 32-Plex Cytokine Panel (Dr. Havrda's Lab) — IHC: GFAP/Iba1 IHC, planimetric counts (Dr. Havrda's Lab) — PK analysis at Takeda |
|
20220528 Matt: Administering tamoxifen to 2 large cohorts now. Have a window allowing us to do in vivo study anytime over the summer. Unused mice can go to NIH studies. We can decide to re-breed in July or so if we appear to be delayed. Western blotting for NLRP3, ASC, Caspase-1, IL-1b work well. We'll double check brain tissues. I don't think mice have IL18? We definitely need more mice, but so far, the combination LPS adn L351P seems to help with P values. | |
| compound |
The only Takeda NLRP3 inhibitor compound we are sending is the one below that has high Kpuu: TR06673219 (tool compound from series 3 that has been discontinued; Holger is in agreement and has given me permission) that is NOT a clinical candidate. We have already agreed with MJJF and Dr. Havrda that any publication will not get initiated unless Takeda is in agreement. We have also clarified this with Matt Russo. No structure will be disclosed to any party and if possible I will also protect the TR number This is a work that is funded by MJFF and we are only providing the compounds : MCC950, Inzomelid and TR06673219. (chemical-structure thumbnail of TR06673219; preserved as body_r03 evidence) |
| meeting 220519 | (veteran 의 ) plasma sample 을 MJF 에 줄거다 : HIGH , INTERM, LOW |
| meeting 220206 | Merck?'s LRRK2 inhibitor caused fibrosis in NHP (within 1m) |
| JF | Brain bank: 150 PD, 5 HC, from Wisconsin parkinson association https://www.wiparkinson.org/ , PMI 정보는 없는 듯, |
| meeting 23012 |
Nanostring on paraffin tissue Wt: ROBUST INHIBITION but tmem CELLS (allele problem?): NO ROBUST INHIBITION, IL-1B no active forms, A35B is a decent allele but not used for this study, in vivo pilot study: 30-50k, optimi: new antibody, used nigericin, to do: mjf internal discussion to revise budget, (Matt: We ordered CST Ab E7V2A, reportedly specific for that cleaved band. Have just enough sample left for one more try. Should be able to match the ELISA and WB data. Once that's working we can optimize with a complete picture. ) → (Conor) After some internal discussion, we agreed that we would like to see the optimization data for the antibody before considering additional funding at this time. Do you know when you might expect to have this data ready to present to us? We would be interested in scheduling another check-in call to review and discuss when it is available. (20230207) i) antibody optimization: not successful (no easy success with the new reagents. Nice bands at the right size but only in LPS/Nigericin control samples collected from larger cultures.) ii) mouse pilot cohort : We can do some basic studies without additional funding while we wait → (main) We're stuck waiting for mice but should be able to make another attempt in the next few weeks. Planning on ramping up the scale so we can generate more materials for these optimization studies. (20230216) Ramp up size (concentration), Reagent (ATP), data recently not quite good with new reagents, could do naïve (?) mice, half a dozen mice, , to make one more (last?) in vitro, don't see bioafctive form band, lose moms (?), not fiving up, seeing some level of inhibition, (20230315) We're still struggling getting he cells in culture. Have all the controls done and awaiting litters from the Tmem mice before running experiments. |
HED
| file | Source | Notes |
|---|---|---|
| (98660) 20230724 Charlie CVB Preliminay human PK for | 1st Atuka human microglia |
EC76: 76% neuroprotection 이었을때 mouse plasma AUC 26,300 ng*hr/ml, 이것은 human plasma AUC 303,000 nM*hr. Then (estimated) EC50: human plasma AUC 100,000 (nM*hr), and human dose >700 mg can achieve this. 100 - 500 mg/day: |
| TR06698660 - July 2023 upload |
- unbound brain concentrations after 100 mg QD were estimated to be above IL-1B IC50 for 24 h. - If IL-1B IC90 coverage for 24 h is desired, then 500 mg QD is estimated |
IHC
- pos need 4-5 micron thickness sections for staining.
- FFPE sections need to be shipped to Alessandra Piersigilli
- zero, ordinal , so no quantification, microglial morphological change. 그래서 안 해 준다고. Ihc 는 ↑ variability, only intracelular (w/o extracellular), only microglia (neuron), then Imperial college 는 보낼 필요? 그들이 slide 만들어주면 (모두 만드는가?)→ 우리가 quantification!
In Vivo strategy
20210805 (RSLT)
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Tamoxifen allele paragraph | Katharine M von Herrmann 2020, PMID | The trailing PMID number is partially redacted/truncated in the source after PMID. |
| HED row 1 | human dose >700 mg can achieve this. | The numeral after > appears to be 700; could read 7000 if a small character is missing. |
| Plan 20220528 row | Holger is in agreement and has given me permission | Holger surname not specified; reading reflects only the first name visible. |
| meeting 23012 row | Wt: ROBUST INHIBITION but tmem CELLS (allele problem?): NO ROBUST INHIBITION, IL-1B no active forms | Highlight wraps across two lines; the comma between fragments is faint. |