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RNA-binding motif 5 (RBM5) is a pro-death tumor suppressor gene in cancer cells. It remains to be determined if it is neurotoxic in the brain or rather if it plays a fundamentally different role in the central nervous system (CNS). Brain-specific RBM5 knockout (KO) mice were given a controlled cortical impact (CCI) traumatic brain injury (TBI). Markers of acute cellular damage and repair were measured in hippocampal homogenates 48 h post-CCI. Hippocampal CA1/CA3 cell counts were assessed 7 days post-CCI to determine if early changes in injury markers were associated with histological outcome. No genotype-dependent differences were found in the levels of apoptotic markers (caspase 3, caspase 6, and caspase 9). However, KO females had a paradoxical increase in markers of pro-death calpain activation (145/150-spectrin and breakdown products [SBDP]) and in DNA repair/survival markers. (pH2A.x and pCREB). CCI-injured male KOs had a significant increase in phosphorylated calcium/calmodulin-dependent protein kinase II (pCaMKII). Despite sex/genotype-dependent differences in KOs in the levels of acute cell signaling targets involved in cell death pathways, 7 day hippocampal neuronal survival did not differ from that of wild types (WTs). Similarly, no differences in astrogliosis were observed. Finally, gene analysis revealed increased estrogen receptor α (ERα) levels in the KO hippocampus in females and may suggest a novel mechanism to explain sex-dimorphic effects on cell signaling. In summary, RBM5 inhibition did not affect hippocampal survival after a TBI in vivo but did modify targets involved in neural signal transduction/Ca(2+) signaling pathways. Findings here support the view that RBM5 may serve a purpose in the CNS that is dissimilar from its traditional pro-death role in cancer.

Supplemental material, JCB878889 Supplemental Material for Robust RBM3 and β-klotho expression in developing neurons in the human brain by Travis C Jackson, Keri Janesko-Feldman, Shaun W Carlson, Shawn E Kotermanski and Patrick M Kochanek in Journal of Cerebral Blood Flow & Metabolism

Extracellular adenosine 3',5'-cyclic monophosphate (3',5'-cAMP) is an endogenous source of localized adenosine production in many organs. Recent studies suggest that extracellular 2',3'-cAMP (positional isomer of 3',5'-cAMP) is also a source of adenosine, particularly in the brain in vivo post-injury. Moreover, in vitro studies show that both microglia and astrocytes can convert extracellular 2',3'-cAMP to adenosine. Here, we examined the ability of primary mouse oligodendrocytes and neurons to metabolize extracellular 2',3'-cAMP and their respective adenosine monophosphates (2'-AMP and 3'-AMP). Cells were also isolated from mice deficient in 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase). Oligodendrocytes metabolized 2',3'-cAMP to 2'-AMP with 10-fold greater efficiency than did neurons (and also more than previously examined microglia and astrocytes); whereas, the production of 3'-AMP was minimal in both oligodendrocytes and neurons. The production of 2'-AMP from 2',3'-cAMP was reduced by 65% in CNPase -/- versus CNPase +/+ oligodendrocytes. Oligodendrocytes also converted 2'-AMP to adenosine, and this was also attenuated in CNPase -/- oligodendrocytes. Inhibition of classic 3',5'-cAMP-3'-phosphodiesterases with 3-isobutyl-1-methylxanthine did not block metabolism of 2',3'-cAMP to 2'-AMP and inhibition of classic ecto-5'-nucleotidase (CD73) with α,β-methylene-adenosine-5'-diphosphate did not attenuate the conversion of 2'-AMP to adenosine. These studies demonstrate that oligodendrocytes express the extracellular 2',3'-cAMP-adenosine pathway (2',3'-cAMP → 2'-AMP → adenosine). This pathway is more robustly expressed in oligodendrocytes than in all other CNS cell types because CNPase is the predominant enzyme that metabolizes 2',3'-cAMP to 2-AMP in CNS cells. By reducing levels of 2',3'-cAMP (a mitochondrial toxin) and increasing levels of adenosine (a neuroprotectant), oligodendrocytes may protect axons from injury.

Macrophages contribute to secondary damage and repair after central nervous system (CNS) injury. Micron-sized paramagnetic iron oxide (MPIO) particles can label macrophages in situ, facilitating three-dimensional (3D) mapping of macrophage accumulation following traumatic brain injury (TBI), via ex vivo magnetic resonance microscopy (MRM) and in vivo monitoring with magnetic resonance imaging (MRI). MPIO particles were injected intravenously (iv; 4.5 mg Fe/Kg) in male C57BL/6J mice (n = 21). A controlled cortical impact (CCI) was delivered to the left parietal cortex. Five protocols were used in naive and injured mice to assess feasibility, specificity, and optimal labeling time. In vivo imaging was carried out at 4.7 Tesla (T). Brains were then excised for 3D MRM at 11.7 T. Triple-label immunofluorescence (MPIO via Dragon Green, macrophages via F480, and nuclei via 4,6-diamidino-2-phenylindole [DAPI]) of brain sections confirmed MPIO particles within macrophages. MRM of naives showed an even distribution of a small number of MPIO-labeled macrophages in the brain. MRM at 48-72 h after CCI and MPIO injection revealed MPIO-labeled macrophages accumulated in the trauma region. When MPIO particles were injected 6 days before CCI, MRM 48 h after CCI also revealed labeled cells at the injury site. In vivo studies of macrophage accumulation by MRI suggest that this approach is feasible, but requires additional optimization. We conclude that MPIO labeling and ex vivo MRM mapping of macrophage accumulation for assessment of TBI is readily accomplished. This new technique could serve as an adjunct to conventional MR approaches by defining inflammatory mechanisms and therapeutic efficacy of anti-inflammatory agents in experimental TBI.