Ciliary Neurotrophic Factor Activates Astrocytes, Redistributes Their Glutamate Transporters GLAST and GLT-1 to Raft Microdomains, and Improves Glutamate HandlingIn Vivo

Abstract

To study the functional role of activated astrocytes in glutamate homeostasis in vivo, we used a model of sustained astrocytic activation in the rat striatum through lentiviral-mediated gene delivery of ciliary neurotrophic factor (CNTF). CNTF-activated astrocytes were hypertrophic, expressed immature intermediate filament proteins and highly glycosylated forms of their glutamate transporters GLAST and GLT-1. CNTF overexpression produced a redistribution of GLAST and GLT-1 into raft functional membrane microdomains, which are important for glutamate uptake. In contrast, CNTF had no detectable effect on the expression of a number of neuronal proteins and on the spontaneous glutamatergic transmission recorded from striatal medium spiny neurons. These results were replicated in vitro by application of recombinant CNTF on a mixed neuron/astrocyte striatal culture. Using microdialysis in the rat striatum, we found that the accumulation of extracellular glutamate induced by quinolinate (QA) was reduced threefold with CNTF. In line with this result, CNTF significantly increased QA-induced [ 18 F]-fluoro-2-deoxyglucose uptake, an indirect index of glutamate uptake by astrocytes. Together, these data demonstrate that CNTF activation of astrocytes in vivo is associated with marked phenotypic and molecular changes leading to a better handling of increased levels of extracellular glutamate. Activated astrocytes may therefore be important prosurvival agents in pathological conditions involving defects in glutamate homeostasis.

Oxidative and excitotoxic insults exert differential effects on spinal motoneurons and astrocytic glutamate transporters: Implications for the role of astrogliosis in amyotrophic lateral sclerosis

“…Both the oxidative and excitotoxic treatments mediated changes in astrocytic phenotype, which were much more prominent following oxidative insult, typical of reactive astrocytes (Pekny and Nilsson, 2005), and likely to improve handling of extracellular Glu (Escartin et al, 2006). The early maintenance of Glu uptake after SIN-1 treatment was not due to either altered overall or cell surface expression of EAAT1/2, indicating astrocytes possess mechanisms to preserve net EAAT activity, and which here was accomplished by a kinetic compensation (increased K m despite decreased V max ) normalizing transport for 2 h. Given the changes in astrocytic phenotype and EAAT distribution, and evidence that GFAP modulates EAAT function and is co-localized with EAAT2 (Hughes et al, 2004;Shobha et al, 2007;Zhou and Sutherland, 2004), the early increase in GFAP may be involved in the initial maintenance of Glu transport in the face of oxidative stressors.…”

Possible role of spinal astrocytes in maintaining chronic pain sensitization: review of current evidence with focus on bFGF/JNK pathway

“…6b). However, two additional potential activators of astrocytes, plasminogen (Liu et al, 2000) and ciliary neurotrophic factor (CNTF) (Escartin et al, 2006) do not cause obvious activation of JNK (Fig. 6b).…”

The Neuroprotective Agent CNTF Decreases Neuronal Metabolites in the Rat Striatum: An in Vivo Multimodal Magnetic Resonance Imaging Study

“…After centrifugation (20,000 g, 15 minutes, 4°C), the supernatant was collected and after fivefold dilution, analyzed by ELISA with the Quantikine ELISA Human CNTF immunoassay kit in reference to standards, according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). 12 Immunohistochemistry Immunohistochemistry was performed on the brain of paraformaldehyde-perfused rats as described previously, 8 with the following antibodies: ED1/CD68 (1:500; Serotec, Raleigh, NC, USA), NeuN (1:2,000; Millipore, Billerica, MA, USA), and vimentin (1:2,000; Calbiochem, La Jolla, CA).…”

“…8,9 Astrocytes overexpressed vimentin in a large part of the striatum injected with lenti-CNTF (28.47 ± 4.80 mm 3 , Figure 1A). …”

“…There was no detectable change in NeuN staining and no immunoreactivity for ED1/CD68, a marker for activated microglia ( Figure 1A), as already reported. 8,9 Ciliary Neurotrophic Factor Induces Multiple Changes in Metabolite Concentrations Rats injected with lenti-LacZ in one striatum and lenti-CNTF in the contralateral striatum were imaged using a 7 T magnet, 1.5 to 3 months after infection. There was no detectable change in T 2 -weighted signal in the striatum for any of the nine rats studied ( Figure 1B).…”