Travis C. Jackson

A mitochondrial pathway for biosynthesis of lipid mediators

The central role of mitochondria in metabolic pathways and in cell death mechanisms requires sophisticated signaling systems. Essential in this signaling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin, are oxidized by the intermembrane space hemoprotein, cytochrome c. We show that an assortment of oxygenated cardiolipin species undergoes phospholipase A2-catalyzed hydrolysis thus generating multiple oxygenated fatty acids, including well known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway including oxidation of polyunsaturated cardiolipins and accumulation of their hydrolysis products – oxygenated linoleic, arachidonic acids and monolyso-cardiolipins – is activated in vivo after acute tissue injury.

Emerging Therapies in Traumatic Brain Injury

Despite decades of basic and clinical research, treatments to improve outcomes after traumatic brain injury (TBI) are limited. However, based on the recent recognition of the prevalence of mild TBI, and its potential link to neurodegenerative disease, many new and exciting secondary injury mechanisms have been identified and several new therapies are being evaluated targeting both classic and novel paradigms. This includes a robust increase in both preclinical and clinical investigations. Using a mechanism-based approach the authors define the targets and emerging therapies for TBI. They address putative new therapies for TBI across both the spectrum of injury severity and the continuum of care, from the field to rehabilitation. They discussTBI therapy using 11 categories, namely, (1) excitotoxicity and neuronal death, (2) brain edema, (3) mitochondria and oxidative stress, (4) axonal injury, (5) inflammation, (6) ischemia and cerebral blood flow dysregulation, (7) cognitive enhancement, (8) augmentation of endogenous neuroprotection, (9) cellular therapies, (10) combination therapy, and (11) TBI resuscitation. The current golden age of TBI research represents a special opportunity for the development of breakthroughs in the field.

The Brain In Vivo Expresses the 2′,3′-cAMP-Adenosine Pathway

J. Neurochem. (2012) 122, 115–125.

Contribution of estrogen receptor subtypes, ERα, ERβ, and GPER1 in rapid estradiol‐mediated enhancement of hippocampal synaptic transmission in mice

Estradiol rapidly modulates hippocampal synaptic plasticity and synaptic transmission; however, the contribution of the various estrogen receptors to rapid changes in synaptic function is unclear. This study examined the effect of estrogen receptor selective agonists on hippocampal synaptic transmission in slices obtained from 3–5‐month‐old wild type (WT), estrogen receptor alpha (ERαKO), and beta (ERβKO) knockout female ovariectomized mice. Hippocampal slices were prepared 10–16 days following ovariectomy and extracellular excitatory postsynaptic field potentials were recorded from CA3‐CA1 synaptic contacts before and following application of 17β‐estradiol‐3‐benzoate (EB, 100 pM), the G‐protein estrogen receptor 1 (GPER1) agonist G1 (100 nM), the ERα selective agonist propyl pyrazole triol (PPT, 100 nM), or the ERβ selective agonist diarylpropionitrile (DPN, 1 µM). Across all groups, EB and G1 increased the synaptic response to a similar extent. Furthermore, prior G1 application occluded the EB‐mediated enhancement of the synaptic response and the GPER1 antagonist, G15 (100 nM), inhibited the enhancement of the synaptic response induced by EB application. We confirmed that the ERα and ERβ selective agonists (PPT and DPN) had effects on synaptic responses specific to animals that expressed the relevant receptor; however, PPT and DPN produced only a small increase in synaptic transmission relative to EB or the GPER1 agonist. We demonstrate that the increase in synaptic transmission is blocked by inhibition of extracellular signal‐regulated kinase (ERK) activity. Furthermore, EB was able to increase ERK activity regardless of genotype. These results suggest that ERK activation and enhancement of synaptic transmission by EB involves multiple estrogen receptor subtypes.

Expression of the 2′,3′-cAMP-Adenosine Pathway in Astrocytes and Microglia

J. Neurochem. (2011) 118, 979–987.

Hemorrhagic Shock Shifts the Serum Cytokine Profile from Pro- to Anti-Inflammatory after Experimental Traumatic Brain Injury in Mice

Secondary insults, such as hemorrhagic shock (HS), worsen outcome from traumatic brain injury (TBI). Both TBI and HS modulate levels of inflammatory mediators. We evaluated the addition of HS on the inflammatory response to TBI. Adult male C57BL6J mice were randomized into five groups (n=4 [naïve] or 8/group): naïve; sham; TBI (through mild-to-moderate controlled cortical impact [CCI] at 5 m/sec, 1-mm depth), HS; and CCI+HS. All non-naïve mice underwent identical monitoring and anesthesia. HS and CCI+HS underwent a 35-min period of pressure-controlled hemorrhage (target mean arterial pressure, 25-27 mm Hg) and a 90-min resuscitation with lactated Ringers injection and autologous blood transfusion. Mice were sacrificed at 2 or 24 h after injury. Levels of 13 cytokines, six chemokines, and three growth factors were measured in serum and in five brain tissue regions. Serum levels of several proinflammatory mediators (eotaxin, interferon-inducible protein 10 [IP-10], keratinocyte chemoattractant [KC], monocyte chemoattractant protein 1 [MCP-1], macrophage inflammatory protein 1alpha [MIP-1α], interleukin [IL]-5, IL-6, tumor necrosis factor alpha, and granulocyte colony-stimulating factor [G-CSF]) were increased after CCI alone. Serum levels of fewer proinflammatory mediators (IL-5, IL-6, regulated upon activation, normal T-cell expressed, and secreted, and G-CSF) were increased after CCI+HS. Serum level of anti-inflammatory IL-10 was significantly increased after CCI+HS versus CCI alone. Brain tissue levels of eotaxin, IP-10, KC, MCP-1, MIP-1α, IL-6, and G-CSF were increased after both CCI and CCI+HS. There were no significant differences between levels after CCI alone and CCI+HS in any mediator. Addition of HS to experimental TBI led to a shift toward an anti-inflammatory serum profile--specifically, a marked increase in IL-10 levels. The brain cytokine and chemokine profile after TBI was minimally affected by the addition of HS.

Extracellular guanosine regulates extracellular adenosine levels

The aim of this investigation was to test the hypothesis that extracellular guanosine regulates extracellular adenosine levels. Rat preglomerular vascular smooth muscle cells were incubated with adenosine, guanosine, or both. Guanosine (30 μmol/l) per se had little effect on extracellular adenosine levels. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) were 0.125 ± 0.020 μmol/l, indicating rapid disposition of extracellular adenosine. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) plus guanosine (30 μmol/l) were 1.173 ± 0.061 μmol/l, indicating slow disposition of extracellular adenosine. Cell injury increased extracellular levels of endogenous adenosine and guanosine, and the effects of cell injury on endogenous extracellular adenosine were modulated by altering the levels of endogenous extracellular guanosine with exogenous purine nucleoside phosphorylase (converts guanosine to guanine) or 8-aminoguanosine (inhibits purine nucleoside phosphorylase). Extracellular guanosine also slowed the disposition of extracellular adenosine in rat preglomerular vascular endothelial cells, mesangial cells, cardiac fibroblasts, and kidney epithelial cells and in human aortic and coronary artery vascular smooth muscle cells and coronary artery endothelial cells. The effects of guanosine on adenosine levels were not mimicked or attenuated by 5-iodotubericidin (adenosine kinase inhibitor), erythro-9-(2-hydroxy-3-nonyl)-adenine (adenosine deaminase inhibitor), 5-aminoimidazole-4-carboxamide (guanine deaminase inhibitor), aristeromycin (S-adenosylhomocysteine hydrolase inhibitor), low sodium (inhibits concentrative nucleoside transporters), S-(4-nitrobenzyl)-6-thioinosine [inhibits equilibrative nucleoside transporter (ENT) type 1], zidovudine (inhibits ENT type 2), or acadesine (known modulator of adenosine levels). Guanosine also increases extracellular inosine, uridine, thymidine, and cytidine, yet decreases extracellular uric acid. In conclusion, extracellular guanosine regulates extracellular adenosine levels.

Role of CNpase in the oligodendrocytic extracellular 2'3'-cAMP-adenosine pathway

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. GLIA 2013;61:1595–1606

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

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.

The nuclear splicing factor RNA binding motif 5 promotes caspase activation in human neuronal cells, and increases after traumatic brain injury in mice

Splicing factors (SFs) coordinate nuclear intron/exon splicing of RNA. Splicing factor disturbances can cause cell death. RNA binding motif 5 (RBM5) and 10 (RBM10) promote apoptosis in cancer cells by activating detrimental alternative splicing of key death/survival genes. The role(s) of RBM5/10 in neurons has not been established. Here, we report that RBM5 knockdown in human neuronal cells decreases caspase activation by staurosporine. In contrast, RBM10 knockdown augments caspase activation. To determine whether brain injury alters RBM signaling, we measured RBM5/10 protein in mouse cortical/hippocampus homogenates after controlled cortical impact (CCI) traumatic brain injury (TBI) plus hemorrhagic shock (CCI+HS). The RBM5/10 staining was higher 48  to 72 hours after injury and appeared to be increased in neuronal nuclei of the hippocampus. We also measured levels of other nuclear SFs known to be essential for cellular viability and report that splicing factor 1 (SF1) but not splicing factor 3A (SF3A) decreased 4  to 72 hours after injury. Finally, we confirm that RBM5/10 regulate protein expression of several target genes including caspase-2, cellular FLICE-like inhibitory protein (c-FLIP), LETM1 Domain-Containing Protein 1 (LETMD1), and amyloid precursor-like protein 2 (APLP2) in neuronal cells. Knockdown of RBM5 appeared to increase expression of c-FLIP(s), LETMD1, and APLP2 but decrease caspase-2.