Neuronal Culture Microenvironments Determine Preferences in Bioenergetic Pathway Use.

In the brain, metabolic supply and demand is directly coupled to neuronal activation. Methods for culturing primary rodent brain cells have come of age and are geared toward sophisticated modeling of human brain physiology and pathology. However, the impact of the culture microenvironment on neuronal function is rarely considered. Therefore, we investigated the role of different neuronal culture supplements for neuronal survival and metabolic activity in a model of metabolic deprivation of neurons using oxygen deprivation, glucose deprivation, as well as live cell metabolic flux analysis. We demonstrate the impact of neuronal culture conditions on metabolic function and neuronal survival under conditions of metabolic stress. In particular, we find that the common neuronal cell culture supplement B27 protects neurons from cell death under hypoxic conditions and inhibits glycolysis. Furthermore, we present data that B27 as well as the alternative neuronal culture supplement N2 restrict neuronal glucose metabolism. On the contrary, we find that the more modern supplement GS21 promotes neuronal energy metabolism. Our data support the notion that careful control of the metabolic environment is an essential component in modeling brain function and the cellular and molecular pathophysiology of brain disease in culture.

Pimecrolimus increases the expression of interferon-inducible genes that modulate human coronary artery cells proliferation.

The pharmacodynamics of the loaded compounds defines clinical failure or success of a drug-eluting device. Various limus derivatives have entered clinics due to the observed positive outcome after stent implantation, which is explained by their antiproliferative activity resulting from inhibition of the cytosolic immunophilin FK506-binding protein 12. Although pimecrolimus also binds to this protein, pimecrolimus-eluting stents failed in clinics. However, despite its impact on T lymphocytes little is known about the pharmacodynamics of pimecrolimus in cultured human coronary artery cells. We were able to show that pimecrolimus exerts antiproliferative activity in human smooth muscle and endothelial cells. Furthermore in those cells pimecrolimus induced transcription of interferon-inducible genes which in part are known to modulate cell proliferation. Modulation of gene expression may be part of an interaction between calcineurin, the downstream target of the pimecrolimus/FK506-binding protein 12-complex, and the toll-like receptor 4. In accordance are our findings showing that silencing of toll-like receptor 4 by siRNA in A549 a lung carcinoma cell line reduced the activation of interferon-inducible genes upon pimecrolimus treatment in those cells. Based on our findings we hypothesize that calcineurin inhibition may induce the toll-like receptor 4 mediated activation of type I interferon signaling finally inducing the observed effect in endothelial and smooth muscle cells. The crosstalk of interferon and toll-like receptor signaling may be a molecular mechanism that contributed to the failure of pimecrolimus-eluting stents in humans.

A functional role of the cyclin-dependent kinase inhibitor 1 (p21(WAF1/CIP1)) for neuronal preconditioning.

Hypoxic preconditioning is thought to rely on gene products regulated by hypoxia-inducible factor (HIF)-1. Here, we show that the HIF-1 target gene cyclin-dependent kinase inhibitor 1, p21(WAF1/CIP1), is essential for neuroprotection by hypoxic/aglycemic or erythropoietin preconditioning using wild-type and p21(WAF1/CIP1)-deficient neurons. Furthermore, overexpression of wild-type p21(WAF1/CIP1) or phospho-mutants significantly increased cell death after hypoxia/aglycemia. Moreover, deferoxamine-induced endogenous tolerance did not involve p21(WAF1/CIP1) expression in cortical neurons. Our data suggest that balanced expression and potentially posttranslational regulation of p21(WAF1/CIP1) is required for hypoxic preconditioning.