LTP expression mediated by autonomous activity of GluN2B-bound CaMKII

May combine two claims.

Our findings in HEK cells confirm prior results with cell-free assays indicating that Ca2+/CaM stimulation is sufficient for the induction of condensation but that pT286 is required for its maintenance beyond the initial stimulus.

“For CaMKII co-condensation with GluN2B, our findings in HEK cells confirm prior results with cell-free assays20,21 indicating that Ca2+/CaM stimulation is sufficient for the induction of condensation but that pT286 is required for its maintenance beyond the initial stimulus.”

• cites [20] Hosokawa, 2021, CaMKII activation persistently segregates postsynaptic proteins via liquid phase separation, Nat. Neurosci. sourceSupported
• cites [21] Cai, 2021, CaMKIIα-driven, phosphatase-checked postsynaptic plasticity via phase separation, Cell Res. sourcePartly supported

  The condensate experiments (Fig. 5, ‘Ca²⁺ oscillation-modulated upper layer PSD assembly formation’; page unavailable) show that Ca²⁺/CaM stimulation promotes CaMKIIα enrichment in GluN2B/PSD-95 droplets. The Results (Fig. 3; page unavailable) show that T286 phosphorylation enhances GluN2B binding and that purified phosphorylated CaMKIIα retains binding after Ca²⁺/CaM removal. They do not directly establish that pT286 is required to maintain the condensates beyond stimulation; in the tested mixture containing PP2A, the condensates dispersed after Ca²⁺ removal. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

LTP induction and expression both require CaMKII binding to GluN2B.

“Specifically, we show that LTP induction and expression both require CaMKII binding to GluN2B”

May combine two claims.

At 30 min after drug addition, neither AS397 nor ruxolitinib affects baseline transmission in wild-type slices.

“At 30 min after drug addition, neither AS397 nor ruxolitinib showed any effect on baseline transmission (Figures 2C and 2D).”

May combine two claims.

AIP inhibits the autonomous activity of GluN2B-bound CaMKII but does not block it completely.

“A different peptide inhibitor of CaMKII, autocamtide-2-related inhibitory peptide (AIP), also inhibited the autonomous activity of GluN2B-bound CaMKII; however, in contrast to tatCN21, it did not block this activity completely.”

May combine two claims.

AS397 and ruxolitinib directly enhance CaMKII binding to GluN2B in vitro and allow CaMKII binding to GluN2B in HEK cells.

“Like AS283, either of these inhibitors directly enhanced CaMKII binding to GluN2B in vitro (Figures 1C and S1A) and allowed CaMKII binding to GluN2B in HEK cells (Figures S1B-S1D).”

May combine two claims.

The autonomous CaMKII activity induced by in vitro binding to immobilized GluN2B C-tail is blocked by ATP-competitive inhibitors AS105, AS283, and AS397 and by tatCN21.

“biochemical kinase activity assays with purified protein showed that the autonomous CaMKII activity that is induced by in vitro binding to the immobilized GluN2B C-tail is blocked not only by the ATP-competitive inhibitors AS105, AS283, and AS397 but also by tatCN21 (Figure 3E).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Co-condensation of CaMKII with GluN2B is not required for LTP maintenance.

“while our study suggests that co-condensation of CaMKII with GluN2B is not required for LTP maintenance”

• AssumptionRests on an assumption: The HEK cell co-condensation assay reflects the regulation of CaMKII-GluN2B co-condensation in neurons/synapses.

May combine two claims.

AS397 and ruxolitinib rescue LTP induction in T286A mutant slices.

“Here, we show such rescue of LTP induction in T286A also by two other ATP-competitive CaMKII inhibitors: AS39724 and ruxolitinib24,25 (Figures 1A and 1B).”

May combine two claims.

AS397 and ruxolitinib effectively reverse LTP in wild-type mice when added 1 min after LTP induction.

“Next, we tested the effects of CaMKII inhibitors on LTP reversal in wild-type mice and found effective LTP reversal by both AS397 and ruxolitinib when added 1 min after LTP induction (Figures 3A and 3B).”

May combine two claims.

AS397 reverses LTP in T286A mice back to baseline, whereas ruxolitinib reduces synaptic strength below baseline.

“Notably, whereas AS397 reversed the LTP in T286A mice back to baseline, ruxolitinib reduced synaptic strength even below baseline (Figures 2A and 2B).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Cells were derived from at least two separate cultures and a minimum of four distinct wells.

“Cells were derived from at least two separate cultures and a minimum of four distinct wells and slices were derived from at least 3 individual animals per group.”

Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

The nature of the experiments and analysis had low potential for introducing bias.

“Blinding was not performed due to resources constraints combined with the nature of experiments and analysis having low potential for introducing bias.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Biological replication was achieved by measuring each unique cell, sample, and hippocampal slice once.

“Biological replication was achieved by measuring each unique cell, sample, and hippocampal slice once.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Post-hoc power analysis confirmed that the studies were adequately powered to detect statistical significance of effects.

“Sample sizes for this study were determined based on previous experience; post-hoc power analysis was conducted, confirming that our studies were adequately powered to detect statistical significance of effects.”

May combine two claims.

The requirement for pT286 in LTP induction but not maintenance helps explain earlier behavioral studies: T286A mice are severely impaired in learning, but when they learn after more extensive training, their memory is normal.

“Notably, this requirement for pT286 in LTP induction but not maintenance helps explain earlier behavioral studies: the T286A mice are severely impaired in learning, but when they learn after more extensive training, their memory is normal.32”

• cites [32] Irvine, 2005, αCaMKII autophosphorylation contributes to rapid learning but is not necessary for memory, Nat. Neurosci. sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Slices were derived from at least 3 individual animals per group.

“Cells were derived from at least two separate cultures and a minimum of four distinct wells and slices were derived from at least 3 individual animals per group.”

Probably not a substantial claim: may not pass S2 (checkable).

The early post-induction requirement for enzymatic CaMKII activity provides an objective definition for the phase of LTP expression.

“As will be discussed, the early post-induction requirement for enzymatic CaMKII activity provides an objective definition for the phase of LTP expression.”

May combine two claims.

Autonomous CaMKII activity mediates post-induction LTP via GluN2B binding, not T286 autophosphorylation.

“Thus, autonomous CaMKII activity mediates post-induction LTP but (1) via GluN2B binding, not T286 autophosphorylation”

Probably not a substantial claim: may not pass S3 (load-bearing).

All comparisons between two groups met parametric criteria.

“All comparisons between two groups met parametric criteria, and independent samples were analyzed using unpaired student’s t-tests.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Randomization was accomplished by reversing the sample order for every experiment.

“Randomization was accomplished by reversing the sample order for every experiment.”

May combine two claims.

Autonomous CaMKII activity mediates post-induction LTP during the intermediary expression phase rather than for long-term maintenance.

“and (2) during the intermediary expression phase rather than for long-term maintenance.”

Probably not a substantial claim: may not pass S3 (load-bearing).

HEK cells were not tested for mycoplasma.

“HEK cells were not tested for mycoplasma.”

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

Blinding was not performed due to resource constraints.

“Blinding was not performed due to resources constraints combined with the nature of experiments and analysis having low potential for introducing bias.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All data were tested for parametric conditions using a Shapiro-Wilk test for normal distribution and a Brown-Forsythe test or an F-test for equal variance.

“All data were tested for their ability to meet parametric conditions, as evaluated by a Shapiro-Wilk test for normal distribution and a Brown-Forsythe test (3 or more groups) or an F-test (2 groups) to determine equal variance.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Investigators were not blinded to the samples during collection or analysis.

“Investigators were not blinded to the samples during collection or analysis.”

May combine two claims.

Later LTP maintenance may still require structural functions of GluN2B-bound CaMKII but not the resulting enzymatic CaMKII activity or their co-condensation.

“Later LTP maintenance may still require structural functions of GluN2B-bound CaMKII but not the resulting enzymatic CaMKII activity or their co-condensation.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Images of individual neurons were acquired from two independent cultures.

“Images of individual neurons from two independent cultures were acquired by 0.5 μm steps over 6 μm.”

• AssumptionRests on an assumption: All GFP-containing constructs used encode EGFP with an additional A206K mutation to minimize dimerization.

Probably not a substantial claim: may not pass S3 (load-bearing).

HEK293 cells were authenticated by STR analysis.

“Human embryonic kidney cells (HEK293; authenticated STR analysis) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Sigma) and 1% penicillin/streptomycin solution (P/S; Gibco).”