D-Serine inhibits non-ionotropic NMDA receptor signaling.

NMDA-type glutamate receptors (NMDARs) are widely recognized as master regulators of synaptic plasticity, most notably for driving long-term changes in synapse size and strength that support learning. NMDARs are unique among neurotransmitter receptors in that they require binding of both neurotransmitter (glutamate) and co-agonist (e.g. d -serine) to open the receptor channel, which leads to the influx of calcium ions that drive synaptic plasticity. Over the past decade, evidence has accumulated that NMDARs also support synaptic plasticity via ion flux-independent (non-ionotropic) signaling upon the binding of glutamate in the absence of co-agonist, although conflicting results have led to significant controversy. Here, we hypothesized that a major source of contradictory results can be attributed to variable occupancy of the co-agonist binding site under different experimental conditions. To test this hypothesis, we manipulated co-agonist availability in acute hippocampal slices from mice of both sexes. We found that enzymatic scavenging of endogenous co-agonists enhanced the magnitude of LTD induced by non-ionotropic NMDAR signaling in the presence of the NMDAR pore blocker, MK801. Conversely, a saturating concentration of d -serine completely inhibited both LTD and spine shrinkage induced by glutamate binding in the presence of MK801. Using a FRET-based assay in cultured neurons, we further found that d -serine completely blocked NMDA-induced conformational movements of the GluN1 cytoplasmic domains in the presence of MK801. Our results support a model in which d -serine inhibits ion flux-independent NMDAR signaling and plasticity, and thus d -serine availability could serve to modulate NMDAR signaling even when the NMDAR is blocked by magnesium. Significance Statement: NMDARs are glutamate-gated cation channels that are key regulators of neurodevelopment and synaptic plasticity and unique in their requirement for binding of a co-agonist (e.g. d -serine) in order for the channel to open. NMDARs have been found to drive synaptic plasticity via non-ionotropic (ion flux-independent) signaling upon the binding of glutamate in the absence of co-agonist, though conflicting results have led to controversy. Here, we found that d -serine inhibits non-ionotropic NMDAR-mediated LTD and LTD-associated spine shrinkage. Thus, a major source of the contradictory findings might be attributed to experimental variability in d -serine availability. In addition, the developmental regulation of d -serine levels suggests a role for non-ionotropic NMDAR plasticity during critical periods of plasticity.

APP fragment controls both ionotropic and non-ionotropic signaling of NMDA receptors.

NMDA receptors (NMDARs) are ionotropic receptors crucial for brain information processing. Yet, evidence also supports an ion-flux-independent signaling mode mediating synaptic long-term depression (LTD) and spine shrinkage. Here, we identify AETA (Aη), an amyloid-ß precursor protein (APP) cleavage product, as an NMDAR modulator with the unique dual regulatory capacity to impact both signaling modes. AETA inhibits ionotropic NMDAR activity by competing with the co-agonist and induces an intracellular conformational modification of GluN1 subunits. This favors non-ionotropic NMDAR signaling leading to enhanced LTD and favors spine shrinkage. Endogenously, AETA production is increased by in vivo chemogenetically induced neuronal activity. Genetic deletion of AETA production alters NMDAR transmission and prevents LTD, phenotypes rescued by acute exogenous AETA application. This genetic deletion also impairs contextual fear memory. Our findings demonstrate AETA-dependent NMDAR activation (ADNA), characterizing AETA as a unique type of endogenous NMDAR modulator that exerts bidirectional control over NMDAR signaling and associated information processing.

Reduced d-serine levels drive enhanced non-ionotropic NMDA receptor signaling and destabilization of dendritic spines in a mouse model for studying schizophrenia.

Schizophrenia is a psychiatric disorder that affects over 20 million people globally. Notably, schizophrenia is associated with decreased density of dendritic spines and decreased levels of d-serine, a co-agonist required for opening of the N-methyl-d-aspartate receptor (NMDAR). We hypothesized that lowered d-serine levels associated with schizophrenia would enhance ion flux-independent signaling by the NMDAR, driving destabilization and loss of dendritic spines. We tested our hypothesis using the serine racemase knockout (SRKO) mouse model, which lacks the enzyme for d-serine production. We show that activity-dependent spine growth is impaired in SRKO mice, but can be acutely rescued by exogenous d-serine. Moreover, we find a significant bias of synaptic plasticity toward spine shrinkage in the SRKO mice as compared to wild-type littermates. Notably, we demonstrate that enhanced ion flux-independent signaling through the NMDAR contributes to this bias toward spine destabilization, which is exacerbated by an increase in synaptic NMDARs in hippocampal synapses of SRKO mice. Our results support a model in which lowered d-serine levels associated with schizophrenia enhance ion flux-independent NMDAR signaling and bias toward spine shrinkage and destabilization.

Destruction complex dynamics: Wnt/ß-catenin signaling alters Axin-GSK3ß interactions in vivo.

The central regulator of the Wnt/ß-catenin pathway is the Axin/APC/GSK3ß destruction complex (DC), which, under unstimulated conditions, targets cytoplasmic ß-catenin for degradation. How Wnt activation inhibits the DC to permit ß-catenin-dependent signaling remains controversial, in part because the DC and its regulation have never been observed in vivo Using bimolecular fluorescence complementation (BiFC) methods, we have now analyzed the activity of the DC under near-physiological conditions in Drosophila By focusing on well-established patterns of Wnt/Wg signaling in the developing Drosophila wing, we have defined the sequence of events by which activated Wnt receptors induce a conformational change within the DC, resulting in modified Axin-GSK3ß interactions that prevent ß-catenin degradation. Surprisingly, the nucleus is surrounded by active DCs, which principally control the degradation of ß-catenin and thereby nuclear access. These DCs are inactivated and removed upon Wnt signal transduction. These results suggest a novel mechanistic model for dynamic Wnt signal transduction in vivo.

Preparation of Precisely Oriented Cryosections of Undistorted Drosophila Wing Imaginal Discs for High Resolution Confocal Imaging.

The combination of immunofluorescence and laser scanning confocal microscopy (LSM) is essential to high-resolution detection of molecular distribution in biological specimens. A frequent limitation is the need to image deep inside a tissue or in a specific plane, which may be inaccessible due to tissue size or shape. Recreating high-resolution 3D images is not possible because the point-spread function of light reduces the resolution in the Z-axis about 3-fold, compared to XY, and light scattering obscures signal deep in the tissue. However, the XY plane of interest can be chosen if embedded samples are precisely oriented and sectioned prior to imaging (Figure 1). Here we describe the preparation of frozen tissue sections of the Drosophila wing imaginal disc, which allows us to obtain high-resolution images throughout the depth of this folded epithelium.