The cyclic AMP cascade is altered in the fragile X nervous system - PubMed (original) (raw)

The cyclic AMP cascade is altered in the fragile X nervous system

Daniel J Kelley et al. PLoS One. 2007.

Abstract

Fragile X syndrome (FX), the most common heritable cause of mental retardation and autism, is a developmental disorder characterized by physical, cognitive, and behavioral deficits. FX results from a trinucleotide expansion mutation in the fmr1 gene that reduces levels of fragile X mental retardation protein (FMRP). Although research efforts have focused on FMRP's impact on mGluR signaling, how the loss of FMRP leads to the individual symptoms of FX is not known. Previous studies on human FX blood cells revealed alterations in the cyclic adenosine 3', 5'-monophosphate (cAMP) cascade. We tested the hypothesis that cAMP signaling is altered in the FX nervous system using three different model systems. Induced levels of cAMP in platelets and in brains of fmr1 knockout mice are substantially reduced. Cyclic AMP induction is also significantly reduced in human FX neural cells. Furthermore, cAMP production is decreased in the heads of FX Drosophila and this defect can be rescued by reintroduction of the dfmr gene. Our results indicate that a robust defect in cAMP production in FX is conserved across species and suggest that cAMP metabolism may serve as a useful biomarker in the human disease population. Reduced cAMP induction has implications for the underlying causes of FX and autism spectrum disorders. Pharmacological agents known to modulate the cAMP cascade may be therapeutic in FX patients and can be tested in these models, thus supplementing current efforts centered on mGluR signaling.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. cAMP production is decreased in FX mice.

Platelets (Control n = 3, FX n = 5) and cortical membranes (n = 3 for each group) from wildtype and fmr1 knockout mice were stimulated with forskolin and levels of cAMP were measured (each n averaged in triplicate). Raw fluorescence (RF) is an inverse index of cAMP levels. The change in cAMP production was assessed using the fractional decrease in RF (FDRF) = (No Forskolin−Forskolin)/(No Forskolin). (a) Mouse platelet RF is comparable in the absence of forskolin [t(6) = 0.99; p = 0.36] but significantly different between groups (GroupxStimulus:[F(1,12) = 7.56;p = 0.018]) in the presence of forskolin [t(6) = −2.62; p = 0.040]. (b) Mouse platelet FDRF shows reduced cAMP induction in FX platelets [t(6) = 3.46; p = 0.014]. (c) Mouse cortex RF is comparable between groups in the absence [t(4) = 1.22;p = 0.29] or presence [t(4) = −1.92;p = 0.13] of forskolin. (d) Mouse cortex FDRF shows reduced FX cAMP induction [t(4) = 3.01; p = 0.04]. Error bars are SEM.

Figure 2

Figure 2. cAMP production is decreased in FX fly.

Head membranes from wildtype, FX, FXR+, FXR++ flies were stimulated with forskolin and levels of cAMP were measured. Raw fluorescence (RF) is an inverse index of cAMP levels. The change in cAMP production was assessed using the fractional decrease in RF (FDRF) = (No Forskolin−Forskolin)/(No Forskolin). (a) RF-based cAMP measures in the fly head (Control n = 10, FX n = 3, FXR+ n = 6; FXR++ n = 6 sets of 20 heads) are comparable between groups in the absence of forskolin [F(3,21) = 1.96; p = 0.15], but not its presence [F(3,21) = 6.23;p = 0.003], since RF intensity in FX flies is larger than the others [t(21) = −4.10;p = 0.000]. (b) Fly head FDRF shows reduced FX cAMP induction relative to control membranes [FDRF:t(21) = 3.85;p = 0.001] and to the FX flies rescued with a single or multiple transgenes [FXR+FDRF:t(21) = 3.23;p = 0.004; FXR++FDRF:t(21) = 4.13;p = 0.000]. Error bars are SEM.

Figure 3

Figure 3. cAMP production is decreased in human FX neural cells.

Unaffected and FX human neural cells were stimulated with forskolin and levels of cAMP were measured. Raw fluorescence (RF) is an inverse index of cAMP levels. The change in cAMP production was assessed using the fractional decrease in RF (FDRF) = (No Forskolin−Forskolin)/(No Forskolin). (a) Neurons (MAP2, green) are present among cells (Hoechst stain, nuclei in blue) in 9 week FX cultures. (b) Extracts of human neural cells were immunoblotted with antibodies to FMRP and actin. FX cells (FX) have reduced levels of FMRP compared to 3 controls (control 1, 2, 3). Similar levels of actin are detected in each sample. (c) Human neural cell RF (2 Controls, each with n = 3; FX, n = 3) is comparable between groups in forskolin's absence [t(6) = 0.82; p = 0.44] but FX cAMP production is lower than controls in the presence of forskolin [RF: t(6) = −3.40; p = 0.015]. (d) Human neural cell FDRF was reduced in FX [t(6) = 3.43; p = 0.01]. Error bars are SEM for FX and pooled SEM for controls. (Pooled SEM)2 = SEM1 2+SEM2 2−2*SEM1*SEM2.

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