Fig 2: C32 and C4 dampen CaMKII activity in Day 1 and Day 3 P19-MN differentiation process. (A,B) Western blot of RA, C32, C4 effect and quantification on endogenous CaMKII activity marked by pThr 286/7 activity in Day 1 P19-differentiated MNs. Day 1 MNs were treated with 1uM atRA, C32, or C4 for 15 min and then harvested for western blot analyses. p = 0.02 (RA), p = 0.005 (C32), and p = 0.003 (C4) determined by paired Student’s t-test (n = 5–7). (C,D) Western blot and quantification of RA, C32, C4 effect on endogenous pCaMKII T286/7 activity in Day 3, P19-MNs. Day 3 MNs were treated with 1uM atRA, C32, or C4 for 30 min and then harvested for western blot analyses. Anti-CRABP1 was used to detect endogenous CRABP1. p= 0.04 (RA), p = 0.003 (C32), and p = 0.002 (C4) determined by paired Student’s t-test (n = 5–7). ß-actin was used as a protein loading control. * p = 0.05, ** p = 0.01. Error bars are presented as mean ± SD.

Fig 3: C32 and C4 dampen CaMKII activity in a CRABP1-dependent manner. (A,B) Western blot and quantification of pCaMKII activity, marked by pThr-286/7 (pCaMKII T286/7) in HEK293T cells treated with DMSO, atRA, C32, or C4 at 0.5–5 µM for 15 min. HEK293T cells were co-transfected with GFP-CaMKII and either empty vector control or CRABP1 expressing vector. Anti-CRABP1 was used to detect CRABP1 in empty vector and CRABP1 transfected samples. p = 0.03 (RA), 0.001 (C32), 0.001 (C4), determined by paired Student’s t-test (n = 4–7). (C,D) Western blot for detecting endogenous CaMKII activity, marked by pThr-286/7, in P19 cells treated with atRA, C32, or C4 at 0.5–5 µM for 15 min. Anti-CRABP1 was used to detect endogenous CRABP1. ß-actin was used as a protein loading control. p < 0.0001 (atRA, C32, and C4) determined by paired Student’s t-test (n = 12–19). * p = 0.05, *** p = 0.001, “n.s.” not significant. Error bars are presented as mean ± SD.

Fig 4: A P19-derived, in vitro MN culture system. (A) Workflow of P19 MN differentiation. First, adherent P19 cells were suspended in +RA (0.5 µM) medium for embryoid body (EB) formation over a two-day period. EB’s were then exchanged into +RA, +Shh medium for neurosphere (NS) formation. Optimal EB and NS formation was achieved through the use of an up-right T75 flask. NS were then dissociated and seeded onto a Matrigel-coated 6-well plate for MN differentiation. Compound experiments were conducted by first depleting atRA and Shh on the relevant day (Day 1 or Day 3) for 18 h. Immediately after the 18-h depletion (indicated by an open circle), MNs were treated with compound at 1 µM for 15 min for Day 1 MNs and 1 µM for 30 min for Day 3 MNs. MNs were then harvested for western blot analyses. (B) Brightfield images of undifferentiated P19 cells (left) and P19-MN differentiated cells on Day 3 (right). Scale bar (white) indicates 100 µm length. (C) qPCR detecting the expression of MN-specific markers, HB9, ChaT, and Isl2. (D) qPCR detecting the expression of Crabp1. Error bars are presented as ± SD. qPCR was conducted in two independent experiments.

Fig 5: CRABP1 dampens CaMKII activity to protect against neurotoxic Ca2+ overload. (A) MTT cell viability assay of WT and CRABP1-MN1 exposed to ionomycin (5 µM, 18 h). Cell viability was measured via formazan generation with an absorbance at 570 nm. p = 0.01 determined by Student’s t-test. MTT assay was conducted in five independent experiments with 3–12 technical replicates per experiment. ** p < 0.01 determined by Student’s t-test. Values are mean ± SD. (B) Cell viability assay of WT MN1 to measure the protective effects of atRA, C32, and C4. WT-MN1 cells were pretreated with atRA, C32, or C4 (0.5–5 µM) for 1.5 h. Immediately after pre-treatment, ionomycin (4 µM) or DMSO (as vehicle control) was added to induce cell death and co-incubated with atRA, C32, or C4. p = 0.34 (RA), p = 0.006 (C32), p = 0.0004 determined with Student’s t-test. (C4) (**** p < 0.0001, DMSO vs. Ionmycin; ## p < 0.01 Ionomycin vs. C32; ### p < 0.001 Ionomycin vs. C4). Three independent experiments were performed. (C) Western blots of CaMKII activity marked by pThr 286/7 in wild-type (WT) and CRABP1 over-expressing MN1 cells treated with medium control, DMSO control, or ionomycin (10 µM, 5–10 min). Beta-actin was used as a loading control. Endogenous and over-expressed 3XFlag-HA-tagged CRABP1 expression was monitored with anti-CRABP1. In order to detect a much lower level of endogenous CRABP1 in WT-MN1, twice as much lysate was loaded for WT-MN1 as compared to the loaded CRABP1-MN1 (over-expression) cell lysate. (D) A model depicting the protective role of CRABP1 in MNs when they are exposed to (1) excitotoxicity which results in (2) pathological increases in [Ca2+]I (purple circles), and subsequent CaMKII over-activation and phosphorylation of Ca2+ permeable AMPA receptors. (3) CRABP1-RA could inhibit pCaMKII over-activation, ultimately protecting cells from AMPA-mediated Ca2+ overload and death (4). The model was illustrated using Biorender.com.