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.


MRI assessment and data analysis
All MR studies were conducted using a 9.4 T MR system (Bruker BioSpin, Billerica, MA, USA) with 440 mT/m gradients and a combination of a linear birdcage resonator (7 cm in diameter) for signal transmission and a 2 × 2 surface coil array for signal detection. An initial MRI study was performed during the occlusion period to measure baseline lesion volumes. Time-of-flight MRA was first performed to confirm that the MCA remained occluded after moving the animal from the surgery bench. MRA images were acquired using a three-dimensional fast low-angle shot (3D-FLASH) sequence with TE = 2.5 ms, TR = 21 ms, FA = 20º, and NA = 1. One slice was recorded (12 mm thick) with FOV = 19.2 × 19.2 mm 2 and a resolution of 0.120 mm/pixel (160 × 160 × 100-point matrix). To measure lesion volumes, ADC maps were obtained 8 minutes after occlusion but before removing the occluding filament. The area of ischemic damage was determined by counting pixels with ADC values below a threshold in the ipsilateral brain hemisphere. ADC values in healthy rat brain normally do not fall below 0.55 × 10 -3 mm 2 /s; therefore, this threshold provides a convenient means of segmenting abnormal tissue. Diffusion-weighted images (DWI) used to calculate ADC maps were acquired using diffusion-tensor imaging with a spin-echo echo-planar imaging sequence (DTI-EPI) with the following parameters: FOV = 24 × 16 mm 2 , matrix = 96 × 64 points (in-plane resolution = 0.25 mm/pixel), 14 consecutive slices (1 mm thick), TE = 27 ms, TR = 4 s, NA = 4, and diffusion b values of 300, 600, 900, 1200, 1600 and 2000 s/mm 2 applied in the z direction. To analyse lesion volume evolution, a multi-slice multi-spin-echo (MSME) sequence was performed with the following acquisition parameters: TE = 9 ms, TR = 3 s, 16 echos, NA = 2, 14 consecutive slices (1 mm thick), FOV = 19.2 × 19.2 mm 2 and an inplane resolution of 0.1 mm/pixel (192 × 192-point matrix). These data were also used to calculate T2 relaxation maps. All images were processed using ImageJ (Rasband WS, National Institutes of Health, Bethesda, MD, USA) on an independent workstation. A treatment-blinded researcher determined infarct volumes from quantitative ADC maps and T2-weighted images averaged between the 4th to 8th echoes. Edema formation was measured using midline deviation (MD) calculated as the ratio between ipsilateral and contralateral hemispheric volumes; lesion volumes were then calculated in mm 3 /MD. Lesion volume as a percentage of the ipsilateral hemispheric volume was calculated as (lesion volume [mm 3 /MD]/ipsilateral hemispheric volume [mm 3 ]) × 100. Images were evaluated by a treatment-blinded researcher.
RBM3 mRNA quantification by qPCR RBM3 mRNA expression was evaluated by quantitative polymerase chain reaction (qPCR) assay. Homogenized sections (around 100 mg) stored at -80ºC were directly submerged in lysis buffer avoiding previous thawing and extracted using a commercial column-based method (PureLinkTM RNA Mini Kit, Invitrogen, USA). Final elution was in 50 μL RNase-free water. RNA quantity and quality were determined by microvolume spectrophotometry (NanoDrop, Thermo Fisher Scientific, USA). To evaluate the purity of RNA extract, the ratios 260:280 and 260:230 were studied, and only samples with ratios over 2 and 1.7 were included in the study. A total amount of 1 μg of RNA was used for retrotranscription into cDNA using the GoScript™ Reverse Transcription System (Promega, USA) and a thermocycler (Biometra, Germany). Briefly, the volume corresponding to 1 μg RNA was diluted in RNase-free water to a total final volume of 11 μL and 1 μL of random primers (0.5 μg in total) was added. This mix was incubated for 5 minutes at 70ºC allowing unwinding of secondary RNA structures before rapid cooling in ice for 10 minutes. Reverse transcription mix was composed of 4 μL reaction buffer (5X), 2 μL of MgCl2 (2.5 mM in the final volume), 1 μL dNTPs (0.5 mM of each nucleotide in the final volume), and 1 μL GoScript™ reverse transcriptase. The final reaction mix, totaling 20 μL, was incubated for 5 minutes at 25ºC to allow annealing, and 60 minutes at 42ºC to allow extension of the cDNA strand. After reverse transcription, the product was diluted in 1:5 in RNase-free water. Finally, RBM3 expression and reference genes β-actin and RPL13 were studied by qPCR using the GoTaq qPCR masterMix (Promega, USA) and Mx3005P qPCR system (Agilent Technologies, USA). Briefly, a total volume of 2 μL diluted cDNA was mixed with 3.5 μL nuclease-free water, 0.5 μL of 10 mM specific primers (0.25 μL for each sense), and 6 μL GoTaq® qPCR Master Mix (2X), to a total final reaction volume of 12 μL. Reactions were performed in triplicate in 96-well plates. Table 1 shows the primer sequences and Table 2 the qPCR thermal cycling protocol. Quantitative PCR results were analyzed using MxPro Mx3005P v4.10. The average of three Ct values obtained for RBM3 in each sample was relativized to the average of three Ct values obtained for β-actin or Rpl13 expression in the three replicates of each sample. Relative expression was then averaged across all samples in each group, and control samples were used as a baseline to normalize expression in the various groups.

RBM3 quantification by western blot
RBM3 expression was quantified by western blot (WB). Cold buffer lysis was added to brain slices in an approximate 10:1 (volume:weight) proportion and the tissue disrupted using a TissueLyser II (Qiagen, Switzerland); lysates were then centrifuged for 30 minutes at 20,000 g and the supernatants collected for WB. Lysate protein content was quantified using the Bradford method (BioRad, Germany). The requisite volume of cell lysate containing a total 30 μg protein was subjected to SDS-PAGE in 12% Criterion™ TGX™ Precast Midi Protein Gel (BioRad, USA) using a fixed voltage of 180V. Proteins were then electrotrasferred to a PVDF membrane (Millipore, Ireland) using a Trans-Blot semi-dry system (Bio-Rad, USA) with a limited voltage of 25V and 180 milliamps for 2 hours. Post-blot membranes were blocked for 45 minutes with 3% bovine serum albumin (BSA) in tris-chloride buffer containing 0.1% Tween 20 (TBST). Once blocked, target proteins were detected by incubation with primary antibodies against RBM3 (rabbit monoclonal anti-RBM3 ab134946, Abcam, UK) and β-actin (rabbit polyclonal anti-βactin, Abcam, UK) diluted 1:1000 and 1:2000 respectively in TBST with 3% BSA. Primary antibodies were incubated overnight at room temperature with agitation, then the membranes were washed three times in PBST to remove excess primary antibody. Secondary antibody was HRP-conjugated goat anti-rabbit IgG (Dako, Denmark) diluted to 1:5000 in TBST with 3% BSA and incubated for 1 hour at room temperature with agitation. Finally, after washing in TBST, HRP activity was revealed with Pierce™ _ECL Western Blotting Substrate (Thermo Fisher, USA) and detected manually with Xray film. WB results were analyzed using ImageJ (Rasband WS, USA) by measuring the mean gray value of protein bands delimited in ROIs. Relative expression of RBM3 to β-actin was calculated for each sample, and each group sample average was normalized to the control.

RBM3 levels in ischemic stroke patients
RBM3 levels were analyzed in two cohorts of IS patients: one from the Clinical Hospital of Santiago de Compostela ('Santiago'), the other from Dr. Josep Trueta Clinical Hospital in Girona ('Girona'), after approval by the respective ethics committees and in accordance with the STROBE Statement. Recruitment ran from January to December 2017. We began by studying 20 patients with acute IS to determine sample size and RBM3 sampling times: levels were analyzed on admission (baseline) and 2, 24 and 72 hours after admission for IS patients. In view of these preliminary results (Supplementary Fig. 2 and 3), we selected RBM3 levels on admission and at 24 hours after stroke onset for the whole sample. Sample size was calculated using EPIDAT statistical software based on the difference of RBM3 values at 24 hours in relation to outcome at 3 months ( Supplementary Fig. 4). Minimum sample size (n=198) was calculated on the basis of 5% alpha and 80% power. Two independent cohorts totaling 301 patients were included in our study, 172 from Santiago and 129 from Girona, of whom 215 were finally eligible (113 and 102, respectively) according to the inclusion and exclusion criteria (Tables 3 and 4). Stroke Unit patients in both university hospitals were treated in accordance with the guidelines of the Spanish Society of Neurology's Cerebrovascular Diseases Study Group. The primary clinical endpoint was hyperthermia, defined as an axillary temperature ≥37.5°C measured by the Stroke Unit nursing staff. An axillary temperature <37.5ºC was defined as normothermia. The highest temperature measured in the first 24 hours was selected as the variable. The second clinical endpoint was 3-month outcome, 'good' being defined as a modified Rankin Scale (mRS) score <3. Stroke severity was assessed using the National Institute of Health Stroke Scale (NIHSS) score at admission, 24, 48 and 72 hours. Both scores were evaluated by internationally accredited neurologists.

RBM3 analysis in human blood samples
Blood samples were collected in test tubes, centrifuged at 3,000 g for 15 minutes, frozen immediately, and stored at -80°C. RBM3 levels were measured using commercial ELISA kits following manufacturer instructions (Biotez RBM3 ELISA). RBM3 was analyzed over the range 31.25 pg/mL to 2000 pg/mL, with a sensitivity of 10 pg/mL. Intra-and inter-assay coefficients of variation were 1.9% and 2.7%, respectively. We defined percentage variation in RBM3 levels in the first 24 hours as  • Full-size WB gels corresponding to the Fig.1b, d and Fig. 2b, c. Complementary to Fig 1A-B