Validation of a System xc– Functional Assay in Cultured Astrocytes and Nervous Tissue Samples

Disruption of the glutamatergic homeostasis is commonly observed in neurological diseases and has been frequently correlated with the altered expression and/or function of astrocytic high-affinity glutamate transporters. There is, however, a growing interest for the role of the cystine-glutamate exchanger system xc– in controlling glutamate transmission. This exchanger is predominantly expressed in glial cells, especially in microglia and astrocytes, and its dysregulation has been documented in diverse neurological conditions. While most studies have focused on measuring the expression of its specific subunit xCT by RT-qPCR or by Western blotting, the activity of this exchanger in tissue samples remains poorly examined. Indeed, the reported use of sulfur- and carbon-radiolabeled cystine in uptake assays shows several drawbacks related to its short radioactive half-life and its relatively high cost. We here report on the elaborate validation of a method using tritiated glutamate as a substrate for the reversed transport mediated by system xc–. The uptake assay was validated in primary cultured astrocytes, in transfected cells as well as in crude synaptosomes obtained from fresh nervous tissue samples. Working in buffers containing defined concentrations of Na+, allowed us to differentiate the glutamate uptake supported by system xc– or by high-affinity glutamate transporters, as confirmed by using selective pharmacological inhibitors. The specificity was further demonstrated in primary astrocyte cultures from transgenic mice lacking xCT or in cell lines where xCT expression was genetically induced or reduced. As such, this assay appears to be a robust and cost-efficient solution to investigate the activity of this exchanger in physiological and pathological conditions. It also provides a reliable tool for the screening and characterization of new system xc– inhibitors which have been frequently cited as valuable drugs for nervous disorders and cancer.

MLC1: A New Calcium-regulated Protein Conferring Calcium Dependence to Volume-regulated Anion Channels (VRAC) in Astrocytes.

MLC1 is a membrane protein highly expressed by brain perivascular astrocytes. Mutations in the MLC1 gene account for megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling, causing cognitive and motor dysfunctions. It has been demonstrated that MLC1 mutations affect the swelling-activated Cl - currents (I Cl,swell ) mediated by volume-regulated anion channel (VRAC) and the consequent regulatory volume decrease (RVD) and lead to abnormal activation of intracellular signaling pathways linked to inflammation/osmotic stress. Despite this knowledge, the MLC1 physiological role and MLC molecular pathogenesis are still elusive. Following the observations that Ca 2+ regulates all the MLC1-modulated processes and that intracellular Ca 2+ homeostasis is altered in MLC1-defective cells, we applied a multidisciplinary approach including biochemistry, molecular biology, video imaging, electrophysiology and proteomic techniques on cultured astrocytes to uncover new Ca 2+ -dependent signaling pathways controlling MLC1 function. Here, we revealed that MLC1 binds the Ca 2+ effector proteins calmodulin (CaM) and Ca 2+ /CaM-dependent protein kinase II (CaMKII) and, as result, changes its assembly, localization and functional properties in response to Ca 2+ changes. Noteworthy, CaM binding to the COOH terminal promotes MLC1 trafficking to the plasma membrane, while CaMKII phosphorylation of the NH 2 -terminal potentiates MLC1 activation of I Cl,swell . Overall, these results revealed that MLC1 is a Ca 2+ -regulated protein linking VRAC function and, possibly, volume regulation to Ca 2+ signaling in astrocytes. These findings open new avenues of investigations aimed at clarifying the abnormal molecular pathways underlying MLC and other diseases characterized by astrocyte swelling and brain edema.

Dynamic expression of homeostatic ion channels in differentiated cortical astrocytes in vitro

The capacity of astrocytes to adapt their biochemical and functional features upon physiological and pathological stimuli is a fundamental property at the basis of their ability to regulate the homeostasis of the central nervous system (CNS). It is well known that in primary cultured astrocytes, the expression of plasma membrane ion channels and transporters involved in homeostatic tasks does not closely reflect the pattern observed in vivo. The individuation of culture conditions that promotes the expression of the ion channel array found in vivo is crucial when aiming at investigating the mechanisms underlying their dynamics upon various physiological and pathological stimuli. A chemically defined medium containing growth factors and hormones (G5) was previously shown to induce the growth, differentiation, and maturation of primary cultured astrocytes. Here we report that under these culture conditions, rat cortical astrocytes undergo robust morphological changes acquiring a multi-branched phenotype that develop gradually during the 2-week period of culturing. The shape changes were paralleled by variations in passive membrane properties and background conductance owing to the differential temporal development of inwardly rectifying chloride (Cl−) and potassium (K+) currents. Confocal and immunoblot analyses showed that morphologically differentiated astrocytes displayed a robust increase in the expression of the inward rectifier Cl− and K+ channels ClC-2 and Kir4.1, respectively, which are relevant ion channels in vivo. Finally, they exhibited a large diminution of the intermediate filaments glial fibrillary acidic protein (GFAP) and vimentin which are upregulated in reactive astrocytes in vivo. Taken together the data indicate that long-term culturing of cortical astrocytes in this chemical-defined medium promotes a quiescent functional phenotype. This culture model could aid to address the regulation of ion channel expression involved in CNS homeostasis in response to physiological and pathological challenges.

Cholesterol Regulates Exosome Release in Cultured Astrocytes

Exosomes are vesicles secreted by various kinds of cells, and they are rich in cholesterol, sphingomyelin (SM), phosphatidylcholine, and phosphatidylserine. Although cellular sphingolipid-mediated exosome release has been reported, the involvement of other lipid components of cell membranes in the regulation of exosome release is poorly understood. Here, we show that the level of exosome release into conditioned media is significantly reduced in cultured astrocytes prepared from apolipoprotein E (ApoE) knock-out mice when compared to those prepared from wild-type (WT) mice. The reduced level of exosome release was accompanied by elevated levels of cellular cholesterol. The addition of cholesterol to WT astrocytes significantly increased the cellular cholesterol levels and reduced exosome release. PI3K/Akt phosphorylation was enhanced in ApoE-deficient and cholesterol-treated WT astrocytes. In contrast, the depletion of cholesterol in ApoE-deficient astrocytes due to treatment with β-cyclodextrin recovered the exosome release level to a level similar to that in WT astrocytes. In addition, the reduced levels of exosome release due to the addition of cholesterol recovered to the control levels after treatment with a PI3K inhibitor (LY294002). The cholesterol-dependent regulation of exosome release was also confirmed by in vivo experiments; that is, exosome levels were significantly reduced in the CSF and blood serum of WT mice that were fed a high-fat diet and had increased cholesterol levels when compared to those in WT mice that were fed a normal diet. These results suggest that exosome release is regulated by cellular cholesterol via stimulation of the PI3K/Akt signal pathway.

APOE4 genotype exacerbates the depression-like behavior of mice during aging through ATP decline

Population-based studies reveal that apolipoprotein E (APOE) ε4 gene allele is closely associated with late-life depression (LLD). However, its exact role and underlying mechanism remain obscure. The current study found that aged apoE4-targeted replacement (TR) mice displayed obvious depression-like behavior when compared with age-matched apoE3-TR mice. Furthermore, apoE4 increased stress-induced depression-like behaviors, accompanied by declines in the hippocampal 5-HT (1A) radioligand [18F] MPPF uptake evidenced by positron emission tomography (PET). In [18F]-fluorodeoxyglucose PET ([18F]-FDG PET) analyses, the FDG uptake in the prefrontal cortex, temporal cortex and hippocampus of apoE4-TR mice significantly declined when compared with that of apoE3-TR mice after acute stress. Further biochemical analysis revealed that ATP levels in the prefrontal cortex of apoE4-TR mice decreased during aging or stress process and ATP supplementation effectively rescued the depression-like behaviors of elderly apoE4-TR mice. In primary cultured astrocytes from the cortex of apoE-TR mice, apoE4, when compared with apoE3, obviously decreased the mitochondrial membrane potential, mitochondrial respiration, and glycolysis in a culture time-dependent manner. Our findings highlight that apoE4 is a potential risk factor of depression in elderly population by impairing the glucose metabolism, reducing ATP level, and damaging mitochondrial functions in astrocytes, which indicates that in clinical settings ATP supplementation may be effective for elderly depression patients with apoE4 carrier.