Increased levels of NMDA receptor NR2A subunits at pre‐ and postsynaptic sites of the hippocampal CA1: An early response to conditional double knockout of presenilin 1 and 2

Abstract

Greater than 90% of familial Alzheimer’s disease (AD) is linked to mutations of presenilin (PS), and the loss of PS function altogether within mouse brains by conditional double knockout of the PS 1 and 2 genes (PS-cDKO) leads to age-dependent emergence of AD phenotypes, including neurodegeneration and reduced synaptic plasticity in the hippocampal CA1. The goal of our study was to identify the ultrastructural and molecular changes at synapses in the hippocampal CA1 of this PS-cDKO mouse model of AD. We examined the asymmetric (excitatory) synapses formed on apical dendrites of CA1 pyramidal neurons at 2 months postnatal, an age when AD-like symptoms emerge but brain morphology, as assessed by light microscopy, is still normal. Our quantitative electron microscopic analyses confirm that PS-cDKO hippocampi at 2 months postnatal do not yet exhibit synapse losses or spine size alterations. However, immunocytochemistry reveals that the same region exhibits a 28% increase in the proportion of spines labeled for the NR2A subunits of NMDA receptors (NMDAR), with a 31% increase specifically at postsynaptic densities and a concomitant reduction of these subunits at nonsynaptic sites within spine heads. In contrast, no change in levels or the distribution pattern of NR2B subunit levels were detected within spine heads. Presynaptically, NR2A levels are elevated at axo-spinous junctions and these may contribute to the timing-dependent, long-term depression. These observations point to an early-onset trapping of NMDAR at synapses that are subtle but may underlie the reduced synaptic plasticity at 2 months of age and excitotoxicity at later stages.

NR2A- and NR2B-NMDA receptors and drebrin within postsynaptic spines of the hippocampus correlate with hunger-evoked exercise

“…The primary antibodies directed against the NR2A and the NR2B subunits of NMDARs were the same ones described previously (Aoki et al 2009a, b). The antibody directed against the NR2A subunit of the NMDA receptor was produced in rabbit and corresponds to the amino acids 1265–1464 of the C-terminus of the rodent subunit.…”

“…Moreover, this antibody does not recognize NR1 or NR2A subunits of the NMDAR (Rinaldi et al 2007). We previously conducted EM-ICC and determined that these antibodies recognize asymmetric synapses but not symmetric synapses (Aoki et al 2009b; Fujisawa and Aoki 2003). This antibody has been used to characterize postnatal changes in the level of NR2B-containing NMDARs at asymmetric synapses of the visual cortex.…”

“…The developmental profile is as described earlier by Western blots (Sheng et al Nature 368, 144–147), namely, there is a greater prevalence within younger forebrain tissue than in mature tissue, thereby differing significantly from the developmental profile of NR2A immunoreactivity (Corson et al 2009). Additional electron microscopic immunocy-tochemical studies have revealed that the NR2A and the NR2B antibodies yield distinct subcellular distributions within single brain regions (Fujisawa and Aoki 2003; Aoki et al 2003, 2009b), indicating that the two antibodies do not cross-react very strongly.…”

“…Therefore, in this study, we determined whether ABA induction evokes changes in the level of NMDARs at axo-spinous excitatory synaptic junctions of CA1 pyramidal neurons in ways that correlate with ABA vulnerability. We used electron microscopic immunocytochemistry (EM-ICC) (Aoki et al 2009a, b) to characterize the level and location of two NMDAR subunits, the NR2A and the NR2B, within dendritic spines of the CA1 pyramidal neurons in stratum radiatum, where dendritic remodeling during adolescence is robust and responsive to ABA induction (Chowdhury et al 2014a, c). Given the known distinctions in function that relate directly to the location of NMDARs (Barria and Malinow 2002; Greer and Greenberg 2008; Hardingham and Bading 2010; Petralia et al 2010; Thomas et al 2006; Tovar and Westbrook 1999), we chose EM over Western blotting to distinguish the location of immunoreactivity at postsynaptic membranes versus nonsynaptic and extra-sy-naptic portions on spines, and also to differentiate NMDAR levels at excitatory E-to-E synapses (which form on spines of pyramidal cells) from the E-to-I synapses (which form on dendritic shafts of inhibitory interneurons).…”

Dysfunctional synapse in Alzheimer's disease – A focus on NMDA receptors

“…A first study in transgenic mice expressing the C-terminal of APP demonstrated that NMDAR protein levels were unchanged compared to control mice (Sandhu et al, 1993). Conversely, recent studies showed that presenilins knock-out leads to an early increase in GluN2A subunit expression at post-synaptic densities with a concomitant reduction at non-synaptic sites before synaptic loss (Aoki et al, 2009). In in vitro studies, the effect of Aβ on NMDARs has been also demonstrated.…”