Cold stress protein RBM3 responds to hypothermia and is associated with good stroke outcome

Abstract

RNA-binding motif protein 3 is a molecular marker of hypothermia that has proved neuroprotective in neurodegenerative disease models. However, its relationship to the well-recognized therapeutic effect of hypothermia in ischaemic stroke had not been studied. In this work, the expression of RNA-binding motif protein 3 was investigated in ischaemic animal models subjected to systemic and focal brain hypothermia, specifically the effects of RNA-binding motif protein 3 silencing and overexpression on ischaemic lesions. Moreover, the association of RNA-binding motif protein 3 levels with body temperature and clinical outcome was evaluated in two independent cohorts of acute ischaemic stroke patients (n = 215); these levels were also determined in a third cohort of 31 patients derived from the phase III EuroHYP-1 trial of therapeutic cooling in ischaemic stroke. The preclinical data confirmed the increase of brain RNA-binding motif protein 3 levels in ischaemic animals subjected to systemic and focal hypothermia; this increase was selectively higher in the cooled hemisphere of animals undergoing focal brain hypothermia, thus confirming the direct effect of hypothermia on RNA-binding motif protein 3 expression, while RNA-binding motif protein 3 up-regulation in ischaemic brain regions led to functional recovery. Clinically, patients with body temperature <37.5°C in the first two cohorts had higher RNA-binding motif protein 3 values at 24 h and good outcome at 3 months post-ischaemic stroke, while RNA-binding motif protein 3 levels in the cooled third cohort tended to exceed those in placebo-treated patients. These results make RNA-binding motif protein 3 a molecular marker associated with the effect of hypothermia in ischaemic stroke and suggest its potential application as a promising protective target.

Keywords: RBM3; hypothermia; neuroprotection; stroke; translational study.

Graphical Abstract

Figure 1

Temporal profile of RBM3 expression after hypothermia. (A) Above: Temporal brain Rbm3 mRNA levels after 4 h of SH (target temperature 32°C) or normothermia (target temperature 37°C). Bottom: Temporal brain RBM3 protein expression after 4 h of SH. (B) Above: Brain Rbm3 mRNA expression in healthy animals under FC conditions for 24 h (FC-24h, target temperature 37°C) or FH for 24 h (FH-24h, target temperature 32°C) followed by 3 h of recovery. Bottom: Brain RBM3 protein expression in healthy animals subjected to FC-24h or FH-24h. Rbm3 mRNA levels were relativized to β-actin then normalized to untreated controls. RBM3 protein expression was relativized to β-actin then normalized to control animal expression. WB bands corresponding to β-actin and RBM3 (37 and 17 kDa, respectively) are shown below. Data are shown as mean ± SEM. **P < 0.01 or ***P < 0.001; using one-way or two-way ANOVA followed by a post hoc Bonferroni test (n = 3/group). FC = focal hypothermia control group; FH = focal hypothermia group; SC = Systemic normothermia control groups; SH = systemic hypothermia group. Full-size WB gels are available in Supplementary material.

Figure 2

Hypothermia reduces ischaemic lesion volume and enhances RBM3 expression. (A) MRI assessments of ischaemic injury. Apparent diffusion coefficient maps were recorded before treatment (during surgical ischaemia induction) and T2 images were acquired after 24 h. Since animals subjected to systemic normothermia or SH were sacrificed 3 h after treatment (7 h after hypothermia induction), no T2 images at 24 h are available for these groups. (B) Above: Brain Rbm3 mRNA levels in ischaemic animals subjected to systemic normothermia (I+SC-4h, target temperature 37°C) or SH (I+SH-4h, target temperature 32°C) followed by 3 h of recovery. Below: RBM3 protein expression in animals subjected to I+SC-4h or I+SH-4h, followed by 3 h of recovery. (C) Above: Brain RBM3 mRNA levels in ischaemic animals subjected to focal normothermia (I+FC-24h, target temperature 37°C) or FH (I+FH-24h, target temperature 32°C) followed by 3 h of recovery; Below: Brain RBM3 protein expression in ischaemic animals subjected to I+FC-24h or I+FH-24h, followed by 3 h of recovery. Rbm3 mRNA levels were relativized to β-actin then normalized to the expression in untreated controls; RBM3 protein expression was relativized to β-actin then normalized to the expression in controls. WB bands corresponding to β-actin and RBM3 (37 and 17 kDa, respectively) are shown below. Data are shown as mean ± SEM. **P < 0.01 or ***P < 0.001; using one-way or two-way ANOVA followed by a post hoc Bonferroni test (n = 3/group). FC = focal hypothermia control group; FH = focal hypothermia group; SC = systemic normothermia control groups; SH = systemic hypothermia group. Full-size WB gels are available in Supplementary material.

Figure 3

Histological analysis of RBM3 in brain tissue. (A) Cortical AAV infection 1 month before ischaemia induction. (B) GFP immunofluorescence with neuronal (NeuN), astrocyte (GFAP) and microglial (Iba1) markers. White arrows represent the co-localization observed only for GFP and NeuN.

Figure 4

Effect of RBM3 expression on neurological ischaemic damage. (A) MRI assessments of ischaemic injury and ischaemic lesion volume in AAV-infected animals. Apparent diffusion coefficient maps were recorded before treatment (during surgical ischaemia induction) and T2 images were acquired after 24 h. White arrows show the needle track after virus injection. (B) Representation of the ischaemic injury determined in the four groups studied. Functional deficit in AAV-infected animals determined by grip (C) and rotarod tests (D) respect to the basal conditions (before ischaemia) and 48 h after ischaemia. RBM3⁺: animals infected with AAV2-CamKII-rat-Rbm3-CamKII-eGFP used to enhance RBM3 expression; RBM3^+/0: control group infected with the same empty vector; RBM3⁻: animals infected with AAV2-GFP-U6-rat-Rbm3-shRNA used to silence RBM3 expression; RBM3^−/0: control group infected with the same empty vector. Data are shown as mean ± SEM (n = 8/group).

Figure 5

RBM3 is associated with a good outcome and is temperature-dependent. (A) RBM3 levels at 24 h and (B) ΔRBM3 in normothermic (<37.5°C) and hyperthermic (≥37.5°C) IS patients. (C) RBM3 levels at 24 h and (D) ΔRBM3 in IS patients with poor and good clinical outcome at 3 months. ΔRBM3 was defined as % variation of RBM3 at 24 h respect to the basal levels.

Figure 6

RBM3 levels in patients treated with hypothermia versus placebo. (A) RBM3 levels across the pre-specified collection time-points: basal (before cooling), 26 h (2 h after starting rewarming) and 72 h after stroke onset. Left panel includes all 31 patients and the right panel the SH group only, comprising patients fulfilling pre-defined criteria for per-protocol analyses (n = 7). (B) Boxplots represent the median (interquartile range) of ΔRBM3 values (%) between baseline and rewarming time-points in the SH and placebo arms (left, all cases; right, only patients fulfilling pre-defined criteria for per-protocol analyses). (C) Correlation between RBM3 levels at rewarming and body temperature at 24 h in all patients (left) and those treated with SH (right).