Ribosome profiling in mouse hippocampus: plasticity-induced regulation and bidirectional control by TSC2 and FMRP.

BACKGROUND: Mutations in TSC2 are the most common cause of tuberous sclerosis (TSC), a disorder with a high incidence of autism and intellectual disability. TSC2 regulates mRNA translation required for group 1 metabotropic glutamate receptor-dependent synaptic long-term depression (mGluR-LTD) and behavior, but the identity of mRNAs responsive to mGluR-LTD signaling is largely unknown. METHODS: We utilized Tsc2+/- mice as a mouse model of TSC and prepared hippocampal slices from these animals. We induced mGluR-LTD synaptic plasticity in slices and processed the samples for RNA-seq and ribosome profiling to identify differentially expressed genes in Tsc2+/- and following mGluR-LTD synaptic plasticity. RESULTS: Ribosome profiling reveals that in Tsc2+/- mouse hippocampal slices, the expression of several mRNAs was dysregulated: terminal oligopyrimidine (TOP)-containing mRNAs decreased, while FMRP-binding targets increased. Remarkably, we observed the opposite changes of FMRP binding targets in Fmr1-/y hippocampi. In wild-type hippocampus, induction of mGluR-LTD caused rapid changes in the steady-state levels of hundreds of mRNAs, many of which are FMRP targets. Moreover, mGluR-LTD failed to promote phosphorylation of eukaryotic elongation factor 2 (eEF2) in TSC mice, and chemically mimicking phospho-eEF2 with low cycloheximide enhances mGluR-LTD in TSC mice. CONCLUSION: These results suggest a molecular basis for bidirectional regulation of synaptic plasticity and behavior by TSC2 and FMRP. Our study also suggests that altered mGluR-regulated translation elongation contributes to impaired synaptic plasticity in Tsc2+/- mice.

Disulfide bond requirements for active Wnt ligands.

Secreted Wnt lipoproteins are cysteine-rich and lipid-modified morphogens that bind to the Frizzled (FZD) receptor and LDL receptor-related protein 6 (LRP6). Wnt engages FZD through protruding thumb and index finger domains, which are each assembled from paired ß strands secured by disulfide bonds and grasp two sides of the FZD ectodomain. The importance of Wnt disulfide bonds has been assumed but uncharacterized. We systematically analyzed cysteines and associated disulfide bonds in the prototypic Wnt3a. Our data show that mutation of any individual cysteine of Wnt3a results in covalent Wnt oligomers through ectopic intermolecular disulfide bond formation and diminishes/abolishes Wnt signaling. Although individual cysteine mutations in the amino part of the saposin-like domain and in the base of the index finger are better tolerated and permit residual Wnt3a secretion/activity, those in the amino terminus, the thumb, and at the tip of the index finger are incompatible with secretion and/or activity. A few select double cysteine mutants based on the disulfide bond pattern restore Wnt secretion/activity. Further, a double cysteine mutation at the index finger tip results in a Wnt3a with normal secretion but minimal FZD binding and dominant negative properties. Our results experimentally validate predictions from the Wnt crystal structure and highlight critical but different roles of the saposin-like and cytokine-like domains, including the thumb and the index finger in Wnt folding/secretion and FZD binding. Finally, we modified existing expression vectors for 19 epitope-tagged human WNT proteins by removal of a tag-supplied ectopic cysteine, thereby generating tagged WNT ligands active in canonical and non-canonical signaling.

Epigenetic control of sexual differentiation of the bed nucleus of the stria terminalis.

The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is larger in volume and contains more cells in male than female mice. These sex differences depend on testosterone and arise from a higher rate of cell death during early postnatal life in females. There is a delay of several days between the testosterone surge at birth and sexually dimorphic cell death in the BNSTp, suggesting that epigenetic mechanisms may be involved. We tested the hypothesis that chromatin remodeling plays a role in sexual differentiation of the BNSTp by manipulating the balance between histone acetylation and deacetylation using a histone deacetylase inhibitor. In the first experiment, a single injection of valproic acid (VPA) on the day of birth increased acetylation of histone H3 in the brain 24 h later. Next, males, females, and females treated neonatally with testosterone were administered VPA or saline on postnatal d 1 and 2 and killed at 21 d of age. VPA treatment did not influence volume or cell number of the BNSTp in control females but significantly reduced both parameters in males and testosterone-treated females. As a result, the sex differences were eliminated. VPA did not affect volume or cell number in the suprachiasmatic nucleus or the anterodorsal nucleus of the thalamus, which also did not differ between males and females. These findings suggest that a disruption in histone deacetylation may lead to long-term alterations in gene expression that block the masculinizing actions of testosterone in the BNSTp.