Signaling Defects Affecting Nerve Cell Growth Identified in Fragile X

Steve Bryson, PhD avatar

by Steve Bryson, PhD |

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Abnormal protein production associated with fragile X syndrome was coupled with altered cell growth that favored fast-growing cell types over those that become nerve cells during early development, a study has shown. 

Blocking a signaling pathway known as PI3K corrected both excess protein production and altered cell growth, the researchers found. This suggests that specific interventions may correct these imbalances and be an effective strategy to rescue impairments in fragile X.

The study, “Cell-type-specific profiling of human cellular models of fragile X syndrome reveal PI3K-dependent defects in translation and neurogenesis,” was published in the journal Cell Reports

Fragile X is caused by mutations in the FMR1 gene, which lead to a loss of the encoded protein called FMRP. This protein acts as a shuttle within cells to transport and regulate molecules called messenger RNA (mRNA), which carry instructions from genes to make proteins. 

FMRP controls the production of several proteins at synapses — the site of close contact between nerve cells that allows them to communicate. The absence of FMRP leads to fragile X characteristics and symptoms

In addition to its role in protein production, emerging evidence suggests that FMRP acts to coordinate growth during early nerve cell development, called neurogenesis. However, how the loss of FMRP-mediated protein production is related to impaired cell growth in the developing human brain remains unclear. 

To investigate further, researchers based at the Emory University School of Medicine in Georgia modeled and studied neurogenesis in fragile X by creating patient-derived nerve cell precursors called neural progenitor cells (NPCs). They also created organoids, which are 3D cell cultures that incorporate key features of the brain. 

The team first generated induced pluripotent stem cells (iPSC) from healthy male (control) and fragile X patient skin cells, then converted them to neural progenitor cells or NPCs. Tests confirmed that NPCs, nerve cells (neurons), and organoids derived from patients lacked FMRP. 

FMRP binds to specific mRNAs and acts to suppress protein production. In fragile X-derived NPCs, the overall protein production was significantly elevated compared with controls, “suggesting that this defect is a direct consequence of the loss of FMRP,” the researchers wrote. 

Next, experiments showed that a signaling pathway upstream of protein production, driven by an enzyme called phosphoinositide 3-kinase (PI3K), was significantly increased in patient-derived NPCs. 

Notably, an analysis of postmortem brain tissue from fragile X patients found, compared with controls, higher levels of a protein called p110-beta, which is known to stimulate the PI3K pathway. 

Blocking this pathway was found to reduce the effect of the protein production (translation) defect in fragile X patient NPCs. That, in turn, supported “a model of disrupted PI3K signaling underlying aberrant translation in human [fragile X] neural cells,” the team wrote. 

Using a system specifically developed for this study called neurOMIP, which simultaneously measured multiple molecular characteristics of cell types transitioning from NPSs into neurons, found the protein-synthesis defect in FMRP-deficient cells is more profound in early, fast-growing (proliferative) cell types compared with later-stage, non-proliferating cell populations. 

Moreover, there was a higher abundance of proliferative cell types in fragile X cultures and fewer non-proliferative cells. The loss of FMRP had more impact on these faster-growing early cells. 

“The striking shift toward proliferative cell fates suggests a role for FMRP in regulating global cellular proliferation in human neural cells,” the researchers wrote. 

Next, the team generated 3D organoids from three independent control and fragile X patient iPSCs and found patients’ organoids had a higher percentage of proliferative cells compared with that of the healthy controls.

Further analysis of organoid gene activity found 218 genes with activity that differed between fragile X cells and controls. Genes with less activity were related to more mature cells with neuron characteristics, whereas genes with higher activity were related to fast growth. 

The team then wondered if blocking the overactive PI3K signaling pathway would correct cell proliferation defects. Treating NPCs with two PI3K blockers normalized the population of proliferative cells relative to controls, “suggesting that increased PI3K in [fragile X] is a key driver of downstream defects in both protein synthesis and cell proliferation in early neural progenitor [precursor] cell populations,” the researchers wrote. 

Finally, a chronic treatment strategy in which cells were treated with a PI3K blocker for 12 days appeared to normalize cell fate in some, but not all, early proliferative cell types. In contrast, PI3K inhibition did not significantly affect the abundance of non-proliferating cell populations. 

“We demonstrate that abnormal protein synthesis in FXS is coupled to altered cellular decisions to favor proliferative over neurogenic cell fates during early development,” the scientists wrote. “Furthermore, pharmacologic inhibition of elevated phosphoinositide 3-kinase (PI3K) signaling corrects both excess protein synthesis and cell proliferation in a subset of patient neural cells.”