Na dependent accumulation selleck chemical Imatinib Mesylate of glutamate was measured in untreated astrocytes and in astrocytes overexpressing MIP 2�� in the presence or absence of MIP 2�� siRNA 1. MIP 2�� expression decreased glutamate uptake by about 45%. Signaling events linking MIP 2�� overexpression to GLT 1 reduction We next used signaling pathway specific inhibitors to determine which pathways mediated the decreases in GLT 1 protein levels. MIP 2�� downregulation of GLT 1 expression Inhibitors,Modulators,Libraries was eliminated by PDTC, LY294002, KT5720, and partially rescued by PD98059, suggesting a role for NF ��B, PI 3 K, PKA, and MEK ERK. MIP 2�� influences glutamate neurotoxicity We used a neuron astrocytes co culture system to measure glutamate neurotoxicity.

Neurons were co cultured with differently treated astro cytes for 4 days and then treated with 50 uM glutamate for 4 hours, after which astrocytes were removed and the neuron survival was measured by LDH release and MTT assays. Glutamate was more toxic to neurons in co culture with MIP 2�� transfected or LPS treated astro cytes than normal control astrocytes, Inhibitors,Modulators,Libraries but MIP 2�� siRNA 1 inhibited this change. Immu Inhibitors,Modulators,Libraries nocytochemistry using a MAP 2 antibody to detect neurons confirmed these results. Thus, MIP 2�� overexpression in astrocytes makes neurons more sensitive to glutamate toxicity by reducing GLT expression and activity. In addition, MIP 2�� did not change the effects of astrocyte conditioned media on neurons exposed to glutamate toxicity, suggesting that MIP 2�� alone did not damage neurons nor make neurons more sensitive to glutamate toxicity directly.

Discussion The precise physiological function of chemokines in the brain has not yet been fully determined. For example, we have reported that a novel CXC chemokine, MIP 2��, is widely and constitutively expressed in normal brain and in EAE mice at the onset and relapse phases, but little is known about Inhibitors,Modulators,Libraries its location Inhibitors,Modulators,Libraries or temporal pro duction. Here, we investigated the expression of MIP 2�� in astrocytes, a major source of chemokines in the brain, in response to stimulation with LPS or TNF, which have been linked to CNS diseases from EAE to AIDS de mentia. Optimal MIP 2�� production occurred when confluent astrocyte cultures were stimulated with 5 ug ml of LPS or 50 ng ml TNF during a 48 hour culture, with peak mRNA occurring after 6 hours of culture.

Thus, MIP 2��, like other cytokines, is pro duced by cells intrinsic to the brain, primarily astrocytes, Ku-0059436 but potentially microglial cells and endothelial cells as well. Chemokines regulate leukocyte trafficking to the CNS, but are also involved in pathophysiological processes other than chemotaxis. Here, we demonstrated for the first time that MIP 2�� overexpression decreases GLT 1 expression and glutamate uptake in primary cortical astrocytes, an effect that could be blocked by MIP 2�� siRNA, but did not affect GLAST expression.