GluN3 subunit expression correlates with increased vulnerability of hippocampus and entorhinal cortex to neurodegeneration in a model of temporal lobe epilepsy

Abstract

Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults that is often refractory to anti-epileptic medication therapy. Neither the pathology nor the etiology of TLE are fully characterized, although recent studies have established that the two are causally related. TLE pathology entails a stereotypic pattern of neuron loss in hippocampal and parahippocampal regions, predominantly in CA1 subfield of the hippocampus and layer 3 of the medial entorhinal area (MEA), deemed hallmark pathological features of the disease. Through this work, we address the contribution of glutamatergic N-methyl-D-aspartate receptors (NMDARs) to the pathology (vulnerability and pattern of neuronal loss), and by extension to the pathophysiology (Ca2+ induced excitotoxicity), by assaying the spatial expression of their subunit proteins (GluN1, GluN2A, GluN2B and GluN3A) in these regions using ASTA (area specific tissue analysis), a novel methodology for harvesting brain chads from hard-to-reach regions within brain slices for Western blotting. Our data suggest gradient expression of the GluN3A subunit along the mid-lateral extent of layer 3 MEA and along the CA1-subicular axis in the hippocampus, unlike GluN1 or GluN2 subunits which are uniformly distributed. Incorporation of GluN3A in the subunit composition of conventional diheteromeric (d-) NMDARs yield triheteromeric (t-) NMDARs which by virtue of their increased selectivity for Ca2+ render neurons vulnerable to excitotoxic damage. Thus, the expression profile of this subunit sheds light on the spatial extent of the pathology observed in these regions and implicates the GluN3 subunit of NMDARs in hippocampal and entorhinal cortical pathology underlying TLE.

Visualizing the triheteromeric N-methyl-D-aspartate receptor subunit composition

“…To validate our imaging data in the MEA, we also assayed the hippocampus where GluN3A expression was established using area specific tissue analysis (ASTA) ( Beesley et al, 2022 ). Consistent with these studies, we found widespread expression of GluN3A protein puncta that colocalized with PSD-95 throughout CA1 to CA3 ( Figure 2A ), although the high magnification used for their visualization precluded quantification of their relative abundance in these subfields.…”

“…By assaying the spatial expression of their subunit proteins (GluN1, GluN2A, GluN2B, and GluN3A) using area-specific tissue analysis (ASTA), a novel methodology for harvesting brain chads from hard-to-reach regions within brain slices for Western blotting, we recently showed that GluN3A was expressed in a gradient along the mid-lateral extent of layer three MEA and along the CA1-subicular axis in the hippocampus, unlike GluN1 and GluN2A which were uniformly distributed. The expression profile of GluN3A defined the “zones of vulnerability” in these regions where there was significant cell loss and neurodegeneration, hallmark features of the disease ( Beesley et al, 2022 ). Thus, the GluN3A expression pattern was indicative of the spatial extent of the pathology in the hippocampus and entorhinal cortex implicating t -NMDARs in TLE pathogenesis.…”

“…As described previously (Beesley et al, 2020a(Beesley et al, , 2022, Sprague-Dawley rats (male, postnatal day 40-90, 160-190 g, N = 4) were deeply anesthetized with urethane (1.5 mg/kg; i.p.) prior to intra-aortal fixation with 4% paraformaldehyde (PFA) in a 0.1 M phosphate buffer solution (PB; pH 7.4; 4 • C) following an initial flush with ice-cold saline (0.9%, 4 • C).…”

“…To obtain visual confirmation of the expression and colocalization of GluN1, GluN2, and GluN3 subunits to make t -NMDARs in native tissue, we immunoassayed individual subunit proteins in acutely cut slices of the rat brain (50 μm thick) with fluorescently tagged antibodies ( Supplementary Table 1 ) and imaged them on a high-resolution confocal microscope. We looked specifically in the medial entorhinal area (MEA) where we had initially characterized the voltage-dependent properties of these receptors using electrophysiology, measured their Ca 2+ permeability ( Beesley et al, 2019 , 2020b ) and confirmed expression of the GluN3A protein and its colocalization with GluN1 and GluN2 (A and/or B) subunits ( Kumar, 2016 ) using coimmunoprecipitation experiments ( Beesley et al, 2019 ) and area specific tissue analysis (ASTA) ( Beesley et al, 2022 ). Additionally, we determined whether these subunit proteins colocalized with PSD-95 or Bassoon, to determine the postsynaptic/presynaptic locus of their expression.…”

Effect of chronic F- exposure on ionotropic glutamate AMPA and NMDA receptors in rat hippocampus

“…The predominance of GluN2B-containing NMDARs with reduced expression of GluN2A was observed in ventral hippocampus and cortex areas of rats in pilocarpine-induced model of epilepsy [69], while increase in the total NMDARs number due to overexpression of GluN1 and GluN2A or GluN2B was revealed after PTZ-induced status epilepticus [70]. Epilepsy-induced neurodegeneration accompanied by incorporation of GluN3A subunits to NMDARs resulted in formation of triheteromeric receptors which possess increased selectivity for Ca 2+ over Na + and make the neurons more susceptible to excitotoxic damage [71,72]. On the other hand, the works [73][74][75]…”