Downstream Regulatory Element Antagonist Modulator Regulates Ca²⁺ Homeostasis and Viability in Cerebellar Neurons

Downregulation of the NCX3 protein in SH-SY5Y cell lines overexpressing DREAM or EFmDREAM. Western blots for DREAM, NCX3, and CREB proteins using whole-cell extracts prepared from mock transfected SH-SY5Y cells, from two SH-SY5Y clones overexpressing DREAM, and from two SH-SY5Y clones overexpressing EFmDREAM. The fold DREAM overexpression as well as the percentage downregulation of the NCX3 protein in stably transfected clones were calculated after densitometric analysis. Expression levels of endogenous CREB were used as a control.

Downregulation of NCX3 mRNA and protein in hippocampus and cerebellum of mice overexpressing EFmDREAM. Quantitative real-time PCR showing the expression levels of EFmDREAM (A) and NCX3 (B), in hippocampus and cerebellum from wild-type (wt) and EFmDREAM transgenic mice. Values were normalized by the content of HPRT mRNA. Asterisks represent statistically significant differences between the means relative to corresponding controls (^**p < 0.01; ^*p < 0.05; Student's t test). C, Western blots using cerebellar membranes from three wild-type mice, three transgenic mice not expressing EFmDREAM in the cerebellum (Tg-31), and three transgenic mice expressing EFmDREAM in the cerebellum (Tg-33) were hybridized with antibodies directed to the NCX3 and NCX2 proteins (100 kDa) and to the NCX1 protein (110 kDa).

Handling of cytosolic free [Ca²⁺]_i in transgenic cerebellar granules. A, B, Fluorimetric analysis of fura-2-loaded cerebellar granules in cultures maintained in high (A; 25 mm) or low (B; 5 mm) extracellular K⁺. In both cases, the Ca²⁺ levels were monitored before inducing their elevation with a depolarizing pulse of 60 mm K⁺. The kinetics of the decrease of the Ca²⁺ level after reestablishment of the basal KCl culturing conditions (either 5 or 25 mm) is shown as the inverse of the slope (1/S) of repolarization. Each time course data represents the mean ± SD of the calcium response recorded from at least 40 neurons. wt, Wild type.

Differential effects of extracellular potassium concentrations on the viability in EFmDREAM transgenic neurons. A, Phase-contrast light micrographs of cerebellar neurons from wild-type (wt) or Tg-33 mice cultured for 3 d in vitro (3 DIV) in media containing either 25 or 5 mm KCl. B, Quantitation of cerebellar granules from wild-type or Tg-33 transgenic mice after 3 d in culture using 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI) staining. The bars represent the mean ± SEM of the total number of DAPI-labeled cells in different fields. Six different fields in four different cultures were counted. ^*p < 0.05; ^***p < 0.001, one-way ANOVA test. C, Quantitation of the viability of transgenic granules estimated indirectly by real-time reverse transcription-PCR of EFmDREAM mRNA in cultured Tg-33 cerebellar granules maintained under depolarizing (25 mm KCl) or basal (5 mm KCl) conditions. The results from freshly isolated cells (FIC) and from cells at 3 and 6 DIV correspond to the mean ± SEM of six experiments using different cultures. Significant differences from control (EFmDREAM expression in FIC) are indicated as ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 (one-way ANOVA).

Induced NCX2 expression restores the viability of transgenic cerebellar granules cultured in high K⁺. A, Quantitative real-time PCR showing the temporal expression pattern of NCX2 mRNA in cultured cerebellar granule cells from wild-type (wt; open bars) or transgenic Tg-33 (black bars) mice cultured in the absence or presence of calcineurin inhibitors (FK506+CsA). ^**p < 0.01 and ^***p < 0.001 (one-way ANOVA). B, Quantitation of cell viability estimated indirectly by real-time PCR of EFmDREAM expression in cerebellar transgenic granules cultured in high K⁺ (black bars) in the absence or presence of FK506 and CsA. Significant differences from nontreated cultures are indicated as ^**p < 0.01 and ^***p < 0.001 (one-way ANOVA). For comparison, the expression of the transgene at different days in vitro (DIV) is shown in low K⁺ culturing conditions (gray bars). FIC, Freshly isolated cells.