Mouse study links fragile X to abnormal protein degradation
But researchers cite need for more studies to investigate the connection
A deficiency in the FMRP protein, the underlying cause of fragile X syndrome (FXS), may affect the system within nerve cells that breaks down unwanted proteins, a mouse study suggests.
Data showed that FMRP, its related protein FXR1, and DLG4 gene’s messenger RNA (mRNA) are involved in this protein breakdown system, called ubiquitin/proteasome system (UPS). mRNA is the molecule derived from DNA that guides protein production.
These molecules were found to interact with each other and suppression of FXR1 or DLG4 impaired the growth of neurites, nerve cell projections used to form complex neuronal circuits.
Further studies are needed to investigate a connection between FMRP, FXR1, Dlg4 mRNA, protein degradation, and nerve cell impairment in FXS, the researchers noted.
The study, “FMRP, FXR1 protein and Dlg4 mRNA, which are associated with fragile X syndrome, are involved in the ubiquitin-proteasome system,” was published in the journal Nature Scientific Reports.
FXS is caused by mutations in the FMR1 gene that leads to a deficiency in FMRP protein, ultimately resulting in cognitive, developmental, and behavioral symptoms. However, the mechanisms by which this deficiency causes FXS remain unclear.
FMRP, highly produced in the brain, is known to bind to a similar protein called FXR1, together controlling protein production by interacting with mRNA molecules. In particular, the FMRP protein “is a carrier of Dlg4 mRNA, binding to and stabilizing the mRNA and regulating” its translation into the DLG4 protein, the researchers wrote.
DLG4 plays a crucial role in nerve cell communication.
Now, a team of researchers in Japan set out to learn more about the potential role of FMRP, FXR1, and DLG4 in UPS. As part of the UPS, unwanted and poorly-made proteins in cells are tagged with a small protein called ubiquitin. Such ubiquitinated proteins then are broken down by the large recycling proteasome complex.
Analyzing protein production
The team first blocked the mRNA molecules of these proteins in lab-grown mouse nerve cells and found that overall protein production was reduced in nerve cells lacking FRX1 and DLG4, but was normal in FMRP-deficient cells.
Suppression of either of these three proteins’ mRNA resulted in increased levels of ubiquitinated proteins, suggesting a boost in UPS and associated protein degradation. Consistently, proteasome activity was increased significantly in FMRP- or FXR1-deficient nerve cells.
These findings suggested that FMRP, FXR1, and DLG4 “are involved in the UPS,” the researchers wrote.
Further experiments showed that DLG4 and FXR1 suppression promotes UPS activity independently of FMRP, and that FXR1 deficiency had a greater impact on ubiquitination than DLG4 deficiency in the absence of FMRP.
In addition, FXR1, like FMRP, was found to interact with Dlg4’s mRNA, but contrary to FRMP, it was present in proteasome complexes, which “strongly suggests that FXR1 protein binds to proteasomes, but FMRP does not,” the team wrote.
Notably, FXR1’s binding to proteasomes was not mediated by its interaction with Dlg4’s mRNA.
Restoring neurite formation
Moreover, nerve cells lacking either of the three proteins showed reduced neurite growth. Adding retinoic acid, a molecule that stimulates neurite growth, restored neurite formation in FRMP-deficient cells, but not in those lacking DLG4 or FXR1.
Neurite growth was reduced strongly when both DLG4 and FXR1 were suppressed in nerve cells. The team also found that when neurite outgrowth was active, Dlg4’s mRNA was present at high levels, but little DLG4 protein was detected. In contrast, Fxr1’s mRNA and protein were detected at high and comparable amounts.
These results suggested that “Dlg4 mRNA itself has a function in neurons during neurite outgrowth,” the researchers wrote.
Lastly, the team investigated the activity of Dlg4 and Fxr1 genes in mice during brain development.
While Dlg4’s mRNA was detected during brain development, only a small amount of DLG4 protein was found in the first few days, after which the levels gradually increased. In turn, the levels of Fxr1’s mRNA and FXR1 protein showed similar levels and trends during brain development.
“These results are consistent with the [cell-based] results and support an important role for Dlg4 mRNA in neurite outgrowth during postnatal brain development,” the team wrote.
“This study has revealed a new role for FMRP, FXR1 and Dlg4 mRNA as a UPS-related factor,” the researchers wrote, adding that “these molecules are known to be involved in diseases and vital functions, but their association with the UPS was unknown.”
“Further studies are needed to determine whether Dlg4 mRNA and FXR1 are involved in [neurite growth] as UPS-related factors,” the team concluded.
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