OGD oxygen-glucose deprivation. Our results suggest that RBM3 limits HI-induced apoptosis overall in vivo and in vitro thus at least partially mediating the protective effects of cooling. As expected, rare global differences of gene expression were seen when comparing RBM3 WT and KO even when we applied a less stringent cutoff condition Supplementary Fig.
S6a - S6c , Supplementary Data 2 and 3. Based on these two independent screening approaches we focused on this IGF as in addition it had been reported to induce niche-dependent proliferation of adult NSPCs 23 , Contra contralateral uninjured side , ipsi ipsilateral injured side , LV lateral ventricle, cc corpus callosum, GCL granular cell layer, NC negative control without primary antibodies.
Asterisks indicate target bands. Therefore we intended to figure out whether the downstream effector IGF2 changes in a niche-dependent manner. Normal mouse immunoglobulin msIgG was served as negative control in immunoprecipitation. All images were captured with the same parameters. LV lateral ventricle, cc corpus callosum, GCL granular cell layer, Contra contralateral uninjured side , ipsi ipsilateral injured side. On the other hand RBM3 promotes neuronal differentiation.
In both cell types, RBM3 inhibits apoptosis. NB neuroblast. While injured neurons lose synaptic connectivity and undergo cell death after HI, counteracting endogenous regenerative processes are activated, leading to neurogenesis and synaptogenesis Therapeutic hypothermia is widely known to protect the brain from HI injury 30 , 31 , but there is limited and inconsistent information as to whether hypothermia promotes or inhibits injury-induced NSPC proliferation and neuronal differentiation or if anti-apoptosis is the key mechanism Here we demonstrate that under physiological conditions, RBM3 is expressed in brain, yet its deficiency has no obvious effect on brain development in vivo, probably due to other unknown compensating mechanisms.
However, under pathological conditions such as HI, RBM3 is indispensable for neuroprotection and post-injury neuroregeneration. Like RBM3, it has been reported to promote the proliferation and suppress the apoptosis of neural stem cells in vitro 32 , Extracellular CIRP levels increase in ischemic stroke models causing massive neuronal damage In contrast, no such deleterious effect has been reported with RBM3, which is therefore considered a safer modulator of post-injury neurogenesis than CIRP. IMP family proteins are widely expressed and play important roles at different stages of development.
As IGF2 has been proved to consolidate and enhance memory, a major hippocampal function 41 , a decrease in IGF2 induced by RBM3 deficiency can be expected to affect memory function after HI injury, and may also contribute to discovered memory loss by RBM3 silencing in a chronic neurodegenerative disease model 7. It is feasible to design a small compound to stabilize the RBM3 protein and maintain its expression at high levels by targeting its typical RNA-recognition motif, in order to promote endogenous neurogenesis, particularly in the hippocampus. Such a compound could serve to treat acute brain injury and chronic disease.
A preliminary successful example has already been developed with the CIRP homologue, although its activity requires further tests In addition, the safety of RBM3-based therapy will need to be carefully evaluated for potential tumorigenesis. As IMP family members and IGF2 are critical in promoting cell proliferation, they maintain stem cell stemness under physiological conditions, but are also thought to favor cancer cell progression in diverse tumor types along with their high expression 28 , Fortunately, clinical studies have revealed that in contrast to its CIRP homologue, high RBM3 expression is associated with favorable outcome in various cancers 6.
Although the underlying mechanisms remain largely unknown, this suggests that RBM3-targeted therapy could be safe to administer in brain disorders. All animal experiments were approved by the veterinary office of Basel city authorization number and and were in accordance with the guidelines on laboratory animals. Tadatsugu Taniguchi University of Tokyo, Japan. Right common carotid artery RCCA was exposed and permanently ligated by electrocauterization and subsequently cut. RCCA was only exposed but not ligated in sham animals. Sham animals were not treated with hypoxia. Subsequent BrdU injection was performed every other day for 7 days.
Mice were sacrificed 7 days or 28 days after HI injury for further analysis. TissueTek and frozen in isopentane. Alexa Fluro dye conjugated secondary antibodies were used in Thermo Fisher. Nuclei were couterstained with DAPI. Information for primary antibodies was listed in Supplementary Data 4. Eukitt mounting medium Fluka was used for mounting slides. Stereological coordinates were identified according to adult mouse brain atlas Animals 7 days after HI injury were used for infarction volume estimation.
The infarction volume estimation method was adapted from elsewhere using the Cavalieri estimator probe The contours of direct infarction areas mm 2 , ipsilateral hemisphere areas mm 2 and contralateral hemisphere areas mm 2 were outlined and calculated by Cavalieri point-counting estimator from Stereo Investigator. The corrected infarction areas were calculated as follows:.
A corrected is the corrected infarction area, A direct is the direct infarction area, A ipsi is ipsilateral hemisphere area, and A contra is contralateral hemisphere area. Double-positive cells were imaged using z-stack function with LSM confocal microscope Zeiss. Total cell number N was estimated with the following calculation formula:.
The density of positive cells in the two neurogenic niches was determined by dividing the total positive cell number to the SVZ or DG volume, respectively. The SVZ or DG volumes mm 3 were estimated on adjacent section series with the method as described above. For non-operating and sham animals, the mean of left and right hemisphere cell numbers was presented for each animal.
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For relative intensity quantification of IGF2, all the images were captured with the same parameters. The signal in lateral ventricle was subtracted as background. Three independent experiments were performed. The whole brain excluding cerebellum and meninges from postnatal day 0 male mice was used for NSPC culture. Cells were maintained as neurospheres in uncoated dishes or as monolayer in poly-L-lysine Sigma coated dishes or well chamber slides LabTek.
To passage neurospheres, 0. Primers sequences were listed in Supplementary Data 4. Brain tissues were further homogenized with Dounce tissue grinder on ice.
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Neurospheres assay was performed to test self-renewal capacity of cultured NSPCs. Primary neurospheres were further digested into single cells and plated at the same density to form secondary neurospheres. Triplicates were included in each group. Cultured cells were subjected to directed differentiation or OGD-induced differentiation. The following primary antibodies were used to identify various differentiated cells: MAP2 for neurons; Dcx for neuroblasts; S for glia cell progenitors and Olig2 for oligodendrocyte progenitor cells OPCs.
All quantifications were performed in triplicates. TUNEL staining was used to test late apoptosis. Samples were analyzed by Western blot. Cerebrospinal fluid CSF samples were collected from cisterna magna immediately before sacrificing animals as described previously NSPC culture media were collected immediately before fixing cells. Culture medium from NSPCs were in five replicates.
All in vitro experiments were repeated at least three times. All in vivo experiments included a minimum of five mice per group. Quantification data were presented in standard error mean SEM. For the quantification of data involving contralateral and ipsilateral sides from the same animal, repeated measures two-way ANOVA was performed, and mixed effects model was applied for further comparison with the sham group. Statistical analysis was performed using GraphPad Prism 8.
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Further information on research design is available in the Nature Research Reporting Summary linked to this article. The source data relating to Figs. Uncropped blots are shown in Supplementary Fig.
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All other raw data are available from the authors upon request. A reporting summary for this article is available as a Supplementary Information file. Hypothermia after Cardiac Arrest Study G. Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest. Heinz, U.
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Outcome and prognosis of hypoxic brain damage patients undergoing neurological early rehabilitation. BMC Res. Shankaran, S.
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