Cold stress protein RBM3 responds to temperature change in an ultra-sensitive manner in young neurons

Abstract

Extremely mild hypothermia to 36.0 °C is not thought to appreciably differ clinically from 37.0 °C. However, it is possible that 36.0 °C stimulates highly sensitive hypothermic signaling mechanism(s) and alters biochemistry. To the best of our knowledge, no such ultra-sensitive pathway/mechanisms have been described. Here we show that cold stress protein RNA binding motif 3 (RBM3) increases in neuron and astrocyte cultures maintained at 33 °C or 36 °C for 24 or 48 h, compared to 37 °C controls. Neurons cultured at 36 °C also had increased global protein synthesis (GPS). Finally, we found that melatonin or fibroblast growth factor 21 (FGF21) augmented RBM3 upregulation in young neurons cooled to 36 °C. Our results show that a 1 °C reduction in temperature can induce pleiotropic biochemical changes by upregulating GPS in neurons which may be mediated by RBM3 and that this process can be pharmacologically mimicked and enhanced with melatonin or FGF21.

Keywords: CIRBP; FGF21; RBM3; global protein synthesis; hypothermia; targeted temperature management.

Fig. 1. Comparison of PVDF Membranes Used to Detect RBM3 by Western Blot

(A) Methods used to equalize factors for comparison of PVDF membranes. Neuron homogenates (i.e. samples 1and 2) were loaded onto a 15-well 4–15% SDS gradient gel in five replicates. PVDF membranes were precisely cut to span the width of 2 lanes and match gel length. Proteins were transferred to all 5 membranes at the same time (in the same tank/cassette). Membranes were processed using the same volume/time incubation in blocking solution, TBS washes, primary antibody, secondary antibody, and ECL detection reagent. Membranes were put inside a single film holder for equivalent film exposures in a dark room. (B) Western blots show comparison of PVDF membranes to capture and/or detect RBM3.

Fig. 2. Mild Hypothermia Increases RBM3 in DIV10-11 Neurons

(A) Timeline of experimental procedures. (B) Western blot showing specificity of antibodies to detect RBM3 and (C) CIRBP in neurons given 33°C hypothermia for 48h. (D) Western blot show increased RBM3 but not CIRBP in primary cortical neurons treated 24h or (E) 48h with hypothermia. (F) Densitometry of protein changes for RBM3 (n=5/group) at 24h and (G) 48h (n=5/group). Multiple comparisons were analyzed by one-way-ANOVA and Newman-Keuls post-hoc. Data were significant at p <.05. (*) indicates post-hoc significant difference compared to 37°C normothermia. (#) indicates post-hoc significant difference comparing UMH to mild hypothermia. Graphs show mean + SEM.

Fig. 3. Mild Hypothermia Increases RBM3 in DIV6 Neurons and Increases Protein Synthesis

(A) Timeline of experimental procedures. (B) 20ug protein was loaded onto gels. Western blot show increased RBM3 in primary cortical neurons treated 48h with hypothermia and subjected to 1h re-warm in conditioned media (C.M.) or (C) fresh neurobasal B27 supplement (NBM/B27). (D) Densitometry of RBM3 protein changes in neurons treated with hypothermia for 48h (n=3/group) and then re-warmed in C.M. or (E) Fresh NBM/B27. Multiple comparisons were analyzed by one-way-ANOVA and Newman-Keuls post-hoc. (F) Western blot shows validation of SUnSET and effect of fresh media exchange on protein translation at 37°C. Negative control (no puromycin; NP) are ARA-C treated DIV6 neuron homogenates. Neuron homogenates incubated with puromycin for 30min stain positive with anti-puromycin antibody indicating level of de novo protein synthesis. (G) Level of puromycin staining in neurons treated with hypothermia and rewarmed 1h in C.M. or (H) NBM/B27. Data were significant at p <.05. (*) indicates post-hoc significant difference compared to 37°C normothermia. (#) indicates post-hoc significant difference comparing UMH to mild hypothermia. Graphs show mean + SEM.

Fig 4. Mild Hypothermia Increases RBM3 in Rat Astrocytes

(A) Timeline of experimental procedures. Western blots (20μg/well) showing eIF2α and RBM3 changes in astrocytes given 37°C, 36°C, or 33°C for hypothermia for (B) 24h and (C) 48h. (D) Densitometry of protein changes for RBM3 (n=3/group) at 48h. Data were transformed to log(Y) for analysis. Multiple comparisons were analyzed by one-way-ANOVA and Newman-Keuls post-hoc. Data were significant at p <.05. (*) indicates post-hoc significant difference compared to 37°C normothermia. (#) indicates post-hoc significant difference comparing UMH to mild hypothermia. Graphs show mean + SEM.

Fig 5. FGF21 and Melatonin augment RBM3 induction in young neurons

(A) Western blots (n=2) show effect of 24h treatment of DIV6 neurons with 5nM FGF21, 100μM melatonin, 1μM T090, 5μM SRT1720, or 1μM AZD1080. (B) Western blots show dose effect of 24h FGF21, melatonin, or T090 at 37°C on RBM3 levels in DIV7 neurons. (C) Western blot shows dose effect of 24h FGF21 at 36°C on RBM3 levels in DIV7 neurons. (D) Western blots show effect of 24h treatment combinations (n=3/group) to induce RBM3 in DIV7 neurons cooled to 36°C. FGF21 was applied at 50nM, melatonin 100μM, and T090 at 1μM. (E) Western blots show effect of 48h treatment combinations to induce RBM3 in DIV26 neurons cooled to 36° or 33°C. FGF21 was applied at 50nM, melatonin 100μM, and T090 at 1μM (n=3/group).