Identification of Fragile X Syndrome Specific Molecular Markers in Human Fibroblasts: A Useful Model to Test the Efficacy of Therapeutic Drugs

Abstract

Fragile X Syndrome (FXS) is the most frequent cause of inherited intellectual disability and autism. It is caused by the absence of the fragile X mental retardation 1 (FMR1) gene product, FMRP, an RNA-binding protein involved in the regulation of translation of a subset of brain mRNAs. In Fmr1 knockout (KO) mice, the absence of FMRP results in elevated protein synthesis in the brain as well as increased signaling of many translational regulators. Whether protein synthesis is also dysregulated in FXS patients is not firmly established. Here, we demonstrate that fibroblasts from FXS patients have significantly elevated rates of basal protein synthesis along with increased levels of phosphorylated mechanistic target of rapamycin (p-mTOR), phosphorylated extracellular signal regulated kinase 1/2 (p-ERK 1/2) and phosphorylated p70 ribosomal S6 kinase 1 (p-S6K1). Treatment with small molecules that inhibit S6K1, and a known FMRP target, phosphoinositide 3-kinase (PI3K) catalytic subunit p110β, lowered the rates of protein synthesis in both control and patient fibroblasts. Our data thus demonstrate that fibroblasts from FXS patients may be a useful in vitro model to test the efficacy and toxicity of potential therapeutics prior to clinical trials, as well as for drug screening and designing personalized treatment approaches.

Therapeutic Strategies in Fragile X Syndrome: From Bench to Bedside and Back

“…Consistent with this role are numerous reports of excess mRNA translation and protein synthesis in animal models of FXS and cells from humans with FXS [61][62][63][64][65][66]. Recent studies suggest that FMRP-mediated repression involves its association with polyribosomes and ribosome stalling or direct interaction with the 80S ribosomal subunit [39,67,68].…”

“…Thus, these molecular defects may serve as biomarkers to evaluate the efficiency of disease-targeting therapies in humans. Potential biomarkers for FXS include altered signaling activity of extracellular signal-regulated kinase (ERK)1/2 and phosphoinositide-3-kinase (PI3K) [64][65][66][186][187][188], or altered protein expression or activity of FMRP targets [e.g., amyloid-β precursor protein (AβPP) or matrix metalloproteinase 9 (MMP-9) [162,189]]. Not all defects in signaling in the mouse model have been reproducible across laboratories.…”

“…In the case of FXS, increased density and immature appearance of dendritic spines were detected in patients and mouse models [171,193], and have been widely used to test therapeutic strategies. Pharmacologic and genetic modification of many molecular targets, including neurotransmitter receptors, [62] Lymphoblastoid cells [64], fibroblasts [66] AMPA receptor internalization Increased [43,62,114] N o Altered cellular signaling Increased [64,111,113] or decreased PI3K/mTOR phosphorylation [115]; increased ERK1/2 phosphorylation [112]; increased GSK3β phosphorylation [116] Lymphoblastoid cells [64], lymphocytes and brain tissue [65], fibroblasts [66] Cellular…”

“…† † Minocycline activates MMP-9 but also has other effects. ‡ ‡ Increased protein synthesis is also rescued in FXS patient cell lines [64,66]. § §…”

Fragile X spectrum disorders

“…Recently Matic et al (2014), showed a global downregulation of the MAPK/ERK pathway and decrease in phosphorylation level of ERK1/2 in the murine Fmr1 KO. However, others show an increase in this system in patient fibroblasts (68). A differential expression of many proteins involved in the p53 pathway, Wnt and calcium signaling was also found and led to postulate that calcium imbalance is part of pathophysiology of FXS (69).…”