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Abstract Objectives Use of cumulative mortality adjusted for case mix in patients with acute myocardial infarction for early detection of variation in clinical practice. Design Observational study. Setting 20 hospitals [...]

In previous studies, we have shown that activation of protein kinase C (PKC) rapidly (within minutes) increases the activity and cell surface expression of the glutamate transporter EAAC1 in two systems that endogenously express this transporter (C6 glioma cells and cocultures of neurons and astrocytes). However, the magnitude of the increase in activity is greater than the increase in cell surface expression. In addition, certain compounds completely block the increase in cell surface expression but only partially attenuate the increase in activity. We hypothesized that PKC increases EAAC1 activity by increasing cell surface expression and catalytic efficiency and that two different subtypes of PKC mediate these effects. To address these hypotheses, the PKC subtypes expressed by C6 glioma cells were identified. Of the PKC subtypes that are activated by phorbol esters, only PKCalpha, PKCdelta, and PKCepsilon were observed. G6976, a compound that blocks PKCalpha at concentrations that do not inhibit PKCdelta or PKCepsilon, partially inhibited the increase in uptake but completely abolished the increase in EAAC1 cell surface expression. The 'G6976-insensitive' increase in activity was not associated with a change in total transporter expression but was associated with an increase in the V(max). Na(+)-dependent glycine transport was not increased, providing indirect evidence that the G6976-insensitive increase in activity was not caused by a change in the Na(+) electrochemical gradient required for activity. Finally, by down-regulating different subtypes of PKC, we found evidence that PKCepsilon mediates the increase in EAAC1 activity that is independent of changes in cell surface expression and found further evidence that PKCalpha mediates the increase in cell surface expression. The potential relationship of the present work with a previously identified role for PKCalpha in certain forms of synaptic plasticity is discussed.

The glial glutamate transporter GLT-1 may be the predominant Na(+)-dependent glutamate transporter in forebrain. Expression of GLT-1 correlates with astrocyte maturation in vivo and increases during synaptogenesis. In astrocyte cultures, GLT-1 expression parallels differentiation induced by cAMP analogs or by coculturing with neurons. Molecule(s) secreted by neuronal cultures contribute to this induction of GLT-1, but little is known about the signaling pathways mediating this regulation. In the present study, we determined whether growth factors previously implicated in astrocyte differentiation regulate GLT-1 expression. Of the six growth factors tested, two [epidermal growth factor (EGF) and transforming growth factor-alpha] induced expression of GLT-1 protein in cultured astrocytes. Induction of GLT-1 protein was accompanied by an increase in mRNA and in the V(max) for Na(+)-dependent glutamate transport activity. The effects of dibutyryl-cAMP and EGF were additive but were independently blocked by inhibitors of protein kinase A or protein tyrosine kinases, respectively. The induction of GLT-1 in both EGF- and dibutyryl-cAMP-treated astrocytes was blocked by inhibitors targeting phosphatidylinositol 3-kinase (PI3K) or the nuclear transcription factor-kappaB. Furthermore, transient transfection of astrocyte cultures with a constitutively active PI3K construct was sufficient to induce expression of GLT-1. These data suggest that independent but converging pathways mediate expression of GLT-1. Although an EGF receptor-specific antagonist did not block the effects of neuron-conditioned medium, the induction of GLT-1 by neuron-conditioned medium was completely abolished by inhibition of PI3K or nuclear factor-kappaB. EGF also increased expression of GLT-1 in spinal cord organotypic cultures. Together, these data suggest that activation of specific signaling pathways with EGF-like molecules may provide a novel approach for limiting excitotoxic brain injury.

The therapy of hematologic malignancies has been revolutionized by the development of therapies using chimeric antigen receptor modified T-cells (CAR-T). However, CAR-T cell therapy is often associated with cytokine release syndrome (CRS), characterized by fevers, hypotension, and hypoxia, as well as immune effector cell-associated neurotoxicity syndrome (ICANS). Severe cases of CRS can result in significant morbidity and mortality. Although disease features such as tumor burden may predict for more severe CRS and ICANS, other clinical features and biomarkers predictive of CRS and ICANS remain lacking, with the exception of measurements of serum cytokines following cell infusion (PMID: 27076371, PMID: 24553386). Experimental models have implicated elaboration of inflammatory cytokines such as IL-1 and IL-6 by monocytes in the pathogenesis of CRS and ICANS (PMID: 29808007, PMID: 29808005). Accordingly, the severity and duration of CRS and ICANS can be mitigated in part by IL-6 receptor blockade with tocilizumab and treatment with corticosteroids such as dexamethasone. Recent work has implicated ascorbate in the regulation of the activity of TET enzymes in hematopoietic cells (PMID: 28825709, PMID: 28823558). Given that TET2 deficiency has been associated with increased elaboration of inflammatory cytokines such as IL-6 and IL-1 by macrophages (PMID 3026882, PMID: 28104796, PMID: 28636844), we reasoned that ascorbate deficiency might predict for more pronounced cytokine release in patients leading to more severe CRS or ICANS.

Acute myeloid leukemia (AML) is initiated and sustained by leukemia stem cells (LSCs) which arise from progenitor cells that do not usually self-renew but become aberrantly self-renewing. It is thought that LSCs gain aberrant self-renewal potential by co-opting molecular and cellular programs from hematopoietic stem cells (HSCs) (PMID: 16862118). HSCs have been shown to require tightly regulated protein synthesis rates, where increased or decreased protein synthesis impairs self-renewal (PMID: 24670665), but it is not known if LSCs share this dependence. We have shown that human LSCs reside in the population of AML cells with the highest levels of CD99 (PMID: 28123069). In RNA-sequencing studies, we found that LSCs with high levels of CD99 are depleted for ribosomal protein transcripts. We thus reasoned that similar to HSCs, LSCs may depend on tightly regulated protein synthesis to self-renew.

Several subtypes of Na+-dependent glutamate transporters have been pharmacologically differentiated in brain tissues. Five distinct cDNA clones that express Na+-dependent glutamate transport activity have been isolated. One goal of the current study was to compare the pharmacological properties of the rat GLT-1 subtype of transporter to those identified previously using rat brain tissues. To accomplish this goal, GLT-1 was stably transfected into two different cell lines that express low levels of endogenous transport activity (MCB and L-M (TK-)). Several clones stably transfected with GLT-1 were isolated. In each cell line, Na+-dependent glutamate transport activity was saturable with similar Km values (19 and 37 microM). The pharmacological properties of GLT-1-mediated transport in these cell lines paralleled those observed for the predominant pharmacology observed in cortical crude synaptosomes. These data are consistent with other lines of evidence that suggest that GLT-1 may be sufficient to explain most of the Na+-dependent glutamate transport activity in cortical synaptosomes. Although recent studies using HeLa cells have suggested that GLT-1 can be rapidly up-regulated by activation of protein kinase C (PKC), modulation of PKC or phosphatase activity had no effect on GLT-1-mediated activity in these transfected cell lines. To determine if GLT-1 regulation by PKC is cell-specific, HeLa cells, which endogenously express the EAAC1 subtype of transporter, were stably transfected with GLT-1. Although EAAC1-mediated activity was increased by activation of PKC, we found no evidence for regulation of GLT-1. Despite the present findings, GLT-1 activity may be regulated by PKC under certain conditions.

Sodium-dependent transport into astrocytes is critical for maintaining the extracellular concentrations of glutamate below toxic levels in the central nervous system. In this study, the expression of the glial glutamate transporters GLT-1 and GLAST was studied in primary cultures derived from cortical tissue. In primary astrocytes, GLAST protein levels were approximately one half of those observed in cortical tissue, but GLT-1 protein was present at very low levels compared with cortical tissue. Maintenance of these astrocytes in medium supplemented with dibutyryl-cAMP (dbcAMP) caused a dramatic change in cell morphology, increased GLT-1 and GLAST mRNA levels approximately 5-fold, increased GLAST protein approximately 2-fold, and increased GLT-1 protein >/=8-20-fold. These increases in protein expression were accompanied by 2-fold increases in the Vmax and Km values for Na+-dependent L-[3H]glutamate transport activity. Although GLT-1 is sensitive to inhibition by dihydrokainate in heterologous expression systems, no dihydrokainate sensitivity was observed in astrocyte cultures that expressed GLT-1. Biotinylation with a membrane-impermeant reagent, separation of the biotinylated/cell surface proteins, and subsequent Western blotting demonstrated that both GLT-1 and GLAST were present at the cell surface. Coculturing of astrocytes with neurons also induced expression of GLT-1, which colocalized with the glial specific marker, glial fibrillary acidic protein. Neurons induced a small increase in GLAST protein. Several studies were performed to examine the mechanism by which neurons regulate expression of the glial transporters. Three different protein kinase A (PKA) antagonists did not block the effect of neurons on glial expression of GLT-1 protein, but the addition of dbcAMP to mixed cultures of neurons and astrocytes did not cause GLT-1 protein to increase further. This suggests that neurons do not regulate GLT-1 by activation of PKA but that neurons and dbcAMP regulate GLT-1 protein through convergent pathways. As was observed with GLT-1, the increases in GLAST protein observed in cocultures were not blocked by PKA antagonists, but unlike GLT-1, the addition of dbcAMP to mixed cultures of neurons and astrocytes caused GLAST protein to increase approximately 2-fold. Neurons separated from astrocytes with a semipermeable membrane increased GLT-1 protein, indicating that the effect of neurons was mediated by a diffusible molecule. Treatment of cocultures with high concentrations of either N-methyl-D-aspartate or glutamate killed the neurons, caused GLT-1 protein to decrease, and caused GLAST protein to increase. These studies suggest that GLT-1 and GLAST protein are regulated independently in astrocyte cultures and that a diffusible molecule secreted by neurons induces expression of GLT-1 in astrocytes.