Effects of the presence and absence of amino acids on translation, signaling, and long‐term depression in hippocampal slices from Fmr1 knockout mice

Abstract

Fragile X syndrome (FXS) is caused by silencing of the FMR1 gene and consequent absence of its protein product, fragile X mental retardation protein (FMRP). FMRP is an RNA‐binding protein that can suppress translation. The absence of FMRP leads to symptoms of FXS including intellectual disability and has been proposed to lead to abnormalities in synaptic plasticity. Synaptic plasticity, protein synthesis, and cellular growth pathways have been studied extensively in hippocampal slices from a mouse model of FXS (Fmr1 KO). Enhanced metabotropic glutamate receptor 5 (mGluR5)‐dependent long‐term depression (LTD), increased rates of protein synthesis, and effects on signaling molecules have been reported. These phenotypes were found under amino acid starvation, a condition that has widespread, powerful effects on activation and translation of proteins involved in regulating protein synthesis. We asked if this non‐physiological condition could have effects on Fmr1 KO phenotypes reported in hippocampal slices. We performed hippocampal slice experiments in the presence and absence of amino acids. We measured rates of incorporation of a radiolabeled amino acid into protein to determine protein synthesis rates. By means of western blots, we assessed relative levels of total and phosphorylated forms of proteins involved in signaling pathways regulating translation. We measured evoked field potentials in area CA1 to assess the strength of the long‐term depression response to mGluR activation. In the absence of amino acids, we replicate many of the reported findings in Fmr1 KO hippocampal slices, but in the more physiological condition of inclusion of amino acids in the medium, we did not find evidence of enhanced mGluR5‐dependent LTD. Activation of mGluR5 increased protein synthesis in both wild type and Fmr1 KO. Moreover, mGluR5 activation increased eIF2α phosphorylation and decreased phosphorylation of p70S6k in slices from Fmr1 KO. We propose that the eIF2α response is a cellular attempt to compensate for the lack of regulation of translation by FMRP. Our findings call for a re‐examination of the mGluR theory of FXS.

Regional rates of brain protein synthesis are unaltered in dexmedetomidine sedated young men with fragile X syndrome: A L-[1-11C]leucine PET study

“…In the in vitro studies on hippocampal slices, animals were overdosed with Nembutal, also a GABA agonist, prior to preparation of hippocampal slices. In our recent studies of animals that did not receive anesthesia prior to preparation of the slices, however, we did not see a statistically significant difference in incorporation of labeled leucine into protein in hippocampal slices between WT and Fmr1 KO mice ( Cooke et al, 2019 ). Our PET data on the effects of propofol-anesthesia ( Bishu et al, 2009 ) and dexmedetomidine-sedation in human subjects indicate that these drugs have no effects on rCPS in controls.…”

Selective inhibition of glycogen synthase kinase 3α corrects pathophysiology in a mouse model of fragile X syndrome

“…However, recent findings suggest that the measured increases in bulk protein synthesis are not necessarily a direct consequence of the loss of translational repression by FMRP. For example, it was found that the transcripts overrepresented on translating ribosomes in hippocampal slices from Fmr1 −/y mice are not FMRP targets (98) and, further, that the biochemical and synaptic phenotypes related to protein synthesis in slices are altered substantially when the aCSF is supplemented with essential amino acids (99). Thus, it cannot be assumed that elevated bulk protein synthesis and protein synthesisdependent LTD measured under conditions that are standard for brain slice electrophysiology are root causes of other disease phenotypes.…”

Detection of immunogenic cell death and its relevance for cancer therapy

“…Alternatively, the inhibition of transcription can be accessed via the immunofluorescence microscopy-based detection of nucleolin and fibrillarin, two proteins that colocalize in the nucleus when RNA synthesis is active, yet can be detected as separate entities when transcription is stalled 85 . Besides laborious methods based on the incorporation of radiolabeled amino acids into nascent proteins 203 , translational proficiency can be assessed by polysome profiling, which is commonly based on the separation of cellular lysates on a sucrose gradient coupled to immunoblotting for ribosomal subunits on the fractions collected therefrom 204 , 205 . It may be difficult, however, to scale up polysome profiling for HCS applications.…”