ABSTRACT
Glycogen synthase kinase-3 (GSK-3) activity regulates multiple signal transduction pathways and is also a key component of the network responsible for maintaining stem cell pluripotency. Genetic deletion of Gsk-3α and Gsk-3ß or inhibition of GSK-3 activity via small molecules promotes stem cell pluripotency, yet the mechanism underlying the role for GSK-3 in this process remains ambiguous. Another cellular process that has been shown to affect stem cell pluripotency is mRNA methylation (m6A). Here, we describe an intersection between these components, the regulation of m6A by GSK-3. We find that protein levels for the RNA demethylase, FTO (fat mass and obesity-associated protein), are elevated in Gsk-3α;Gsk-3ß-deficient mouse embryonic stem cells (ESCs). FTO is normally phosphorylated by GSK-3, and MS identified the sites on FTO that are phosphorylated in a GSK-3-dependent fashion. GSK-3 phosphorylation of FTO leads to polyubiquitination, but in Gsk-3 knockout ESCs, that process is impaired, resulting in elevated levels of FTO protein. As a consequence of altered FTO protein levels, mRNAs in Gsk-3 knockout ESCs have 50% less m6A than WT ESCs, and m6A-Seq analysis reveals the specific mRNAs that have reduced m6A modifications. Taken together, we provide the first evidence for how m6A demethylation is regulated in mammalian cells and identify a putative novel mechanism by which GSK-3 activity regulates stem cell pluripotency.
Subject(s)
Adenosine/analogs & derivatives , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism , Glycogen Synthase Kinase 3/physiology , Mouse Embryonic Stem Cells/metabolism , RNA, Messenger/metabolism , Adenosine/metabolism , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Animals , Cells, Cultured , Methylation , Mice , Mice, Knockout , Mouse Embryonic Stem Cells/cytology , Phosphorylation , RNA, Messenger/genetics , UbiquitinationABSTRACT
OBJECTIVE: Fatigue is a common and debilitating symptom of Parkinson's disease (PD) with no evidence-based treatments. While several fatigue scales are partially validated in PD the minimal clinically important difference (MCID) is unknown for any scale but is an important psychometric value to design and interpret therapeutic trials. We thus sought to determine the MCID for the Modified Fatigue Impact Scale (MFIS). METHODS: This is a secondary data analysis from 94 PD participants in an acupuncture trial for PD fatigue. Standard psychometric approaches were used to establish validity and an anchor-based approach was used to determine the MCID. RESULTS: The MFIS demonstrated good concurrent validity with other outcome measures and high internal consistency. MCIDs values were found to be 13.8, 6.8 and 6.2 for the MFIS total, MFIS cognitive, and MFIS physical subscores respectively. CONCLUSIONS: The MFIS is a valid multidimensional measure of fatigue in PD with demonstrable MCID.
