Endogenous nmda receptor subtypes in brain tissue and their assembly and structure

Abstract

The present application relates to endogenous NMDA receptor subtypes in brain tissue and their assembly and structure. The present application provides a method for evaluating the function of endogenous NMDA receptors in brain tissue (cortex and hippocampus), comprising obtaining data on the structure and / or quantity of endogenous NMDA receptors (NMDAR) in the brain tissue, wherein the NMDA receptor comprises a GluN1-N2A-N2B triheteromer; and comparing the structure and / or quantity of the GluN1-N2A-N2B triheteromer with a reference structure and / or quantity reference value, thereby giving an evaluation result of the function of endogenous NMDA receptors in brain tissue. The present application also provides a device for performing the evaluation and a method for drug screening based on changes in the structure and / or quantity of specific NMDA receptors.

Description

Technical Field

[0001] This invention belongs to the field of bioanalysis, and more specifically, this invention relates to endogenous NMDA receptor subtypes and their assembly and structure in brain tissue. Background Technology

[0002] The N-methyl-D-aspartate receptor (NMDAR) is a highly calcium-permeable channel whose opening requires the removal of voltage-dependent magnesium blockade and the binding of co-agonists glycine and glutamate. These combined biophysical properties enable endogenous NMDARs to act as detectors, precisely mediating developmental and activity-dependent synaptic plasticity, particularly in higher brain structures involved in cognitive function, such as the prefrontal cortex and hippocampus. NMDARs are diisotetramers or triisotetramers comprising two GluN1 subunits and two GluN2 (N2A-D) and / or GluN3 (N3A and N3B) subunits. The subunit composition of NMDARs responds differently to synaptic signals at different developmental stages and in different brain regions; this is referred to as a developmental switch, ranging from the dominant GluN2B receptor in newborns to the dominant GluN2A receptor in adults.

[0003] Current research has used heterologous genes to overexpress NMDA receptors in eukaryotic cell lines, and the structures of each subunit have been resolved. However, the expression of endogenous NMDA receptor subunits and receptor assembly in the real brain are strictly regulated. Among the many possible assembly methods, it is still unknown which subunit assembly truly regulates learning and memory, and the proportion of various assembly structures also needs further investigation.

[0004] Therefore, further research is urgently needed on the subunit assembly structure of NMDA receptors in order to gain a deeper understanding of their physiological functions and provide molecular insights into the NMDAR-dependent mechanism behind prominent plasticity. Summary of the Invention

[0005] The purpose of this invention is to provide an endogenous NMDA receptor subtype in brain tissue and its assembly and structure.

[0006] In a first aspect of the invention, a method is provided for assessing the function of endogenous NMDA receptors in brain tissue selected from the group consisting of the cortex, hippocampus, or combinations thereof.

[0007] The method includes the following steps:

[0008] (a) Obtaining data on the structure and / or quantity of endogenous NMDA receptors (NMDARs) in the brain tissue of a subject, wherein the NMDA receptors include GluN1-N2A-N2B triheteromers; and

[0009] (b) The structure and / or quantity of the GluN1-N2A-N2B triheteromeric molecule are compared with reference structures and / or quantity values ​​to provide an evaluation of the function of endogenous NMDA receptors in brain tissue.

[0010] In another preferred embodiment, the evaluation results include: an evaluation of cognitive function, an assessment of mental state, an evaluation of memory function, or a combination thereof.

[0011] In another preferred embodiment, the brain tissue includes brain tissue from childhood or adulthood.

[0012] In another preferred embodiment, the brain tissue is that of an adult.

[0013] In another preferred embodiment, in step (a), the NMDA receptor further comprises: GluN1-N2A, GluN1-N2B, or a combination thereof.

[0014] In another preferred embodiment, in steps (a) and (b), data are preferentially acquired and analyzed according to the following weights: GluN1-N2A-N2B, GluN1-N2A, GluN1-N2B.

[0015] In another preferred embodiment, in steps (a) and (b), data are acquired and analyzed only for the following NMDA receptors: GluN1-N2A-N2B, GluN1-N2A, and GluN1-N2B.

[0016] In another preferred embodiment, the quantity is selected from the group consisting of: the level of NMDAR in the brain tissue, the relative level R (or relative percentage P) of a single NMDAR in the brain tissue, or a combination thereof.

[0017] In another preferred embodiment, the relative level R or relative percentage P of the single NMDAR is calculated according to formula Q1 or Q2:

[0018] R = Zx / Ztotal (Q1)

[0019] P = Zx / Ztotal × 100% (Q2)

[0020] In the formula,

[0021] R represents the relative level of the single NMDAR;

[0022] P is the relative percentage of the single NMDAR;

[0023] Zx represents the level of the single NMDAR in the brain tissue;

[0024] Ztotal represents the total level of all NMDARs in the brain tissue described.

[0025] In another preferred embodiment, the single NMDAR is GluN1-N2A-N2B.

[0026] In another preferred embodiment, Ztotal = Z1 + Z2 + Z3, wherein Z1, Z2 and Z3 are the levels of GluN1-N2A-N2B, GluN1-N2A and GluN1-N2B in the brain tissue, respectively.

[0027] In another preferred embodiment, the quantity reference value includes a reference content.

[0028] In another preferred embodiment, step (b) further includes comparing the relative levels of GluN1-N2A-N2B, GluN1-N2A, and GluN1-N2B with reference contents as follows: GluN1-N2A-N2B 45±3%, GluN1-N2A 35±2%, and GluN1-N2B 20±2%.

[0029] In another preferred embodiment, the reference content is as follows: GluN1-N2A-N2B 45±1%, GluN1-N2A 35±1%, and GluN1-N2B 20±1%.

[0030] In another preferred embodiment, the sum of the reference contents of the three NMDA receptors is approximately 100% (e.g., 99-100%).

[0031] In another preferred embodiment, in step (b), if the relative percentage Pc of GluN1-N2A-N2B is significantly lower than the reference content (or standard value Pc0) of GluN1-N2A-N2B by 45%, it indicates that the content of GluN1-N2A-N2B in the brain tissue of the subject is abnormal, or indicates that the function of GluN1-N2A-N2B in the brain tissue of the subject is decreased; or it indicates a decline in brain function selected from the group consisting of: cognitive function, mental state, memory function, or a combination thereof.

[0032] In another preferred embodiment, in step (b), if the relative percentage Pa of GluN1-N2A is significantly higher than the reference content (or standard value Pa0) of GluN1-N2A by 35%, it indicates that there is an abnormality in the content of GluN1-N2A in the brain tissue of the subject, or indicates a decline in the function of GluN1-N2A-N2B in the brain tissue of the subject; or indicates a decline in brain function selected from the group consisting of: cognitive function, mental state, memory function, or a combination thereof.

[0033] In another preferred embodiment, in step (b), if the relative percentage Pb of GluN1-N2B is significantly higher than the reference content (or standard value Pb0) of GluN1-N2B by 20%, it indicates that there is an abnormality in the content of GluN1-N2B in the brain tissue of the subject, or indicates a decline in the function of GluN1-N2A-N2B in the brain tissue of the subject; or a decline in brain function selected from the group consisting of: cognitive function, mental state, memory function, or a combination thereof.

[0034] In another preferred embodiment, step (b), comparing the structure of the GluN1-N2A-N2B triheteromer with a reference structure, includes:

[0035] (i) Compare the centroid distance (COM) (preferably, compare the COM distance between GluN1-R1 and GluN2-R1, between GluN2-R1 and GluN2-R2, and / or the rotation angle between the two leaflets of GluN2-R2);

[0036] (ii) Compare the structural changes of the GluN1-N2A-N2B triheteromer in the presence of NMDA receptor allosteric modulators;

[0037] (iii) Compare the structural features of the LBD intradimer interface and the LBD interdimer interface;

[0038] (iv) Compare the conformational changes of the GluN2B subunit between GluN1-N2B and GluN1-N2A-N2B;

[0039] (v) Compare the conformational changes of the GluN2A subunit between GluN1-N2A and GluN1-N2A-N2B.

[0040] In another preferred embodiment, (iv), the following structural features are compared: the detwisting of GluN2B-NTD, the tightness of the formation of GluN1-N2B NTD and LBD heterodimers through the R1-R1 and D1-D1 interfaces, the degree of proximity of GluN1(C)-M3 to GluN2B-M3, or combinations thereof.

[0041] In another preferred embodiment, the allosteric modifier comprises: fenfenadil or a pharmaceutically acceptable salt thereof.

[0042] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0043] In another preferred embodiment, the objects include humans and non-human mammals.

[0044] In another preferred embodiment, the objects include humans and non-human primates.

[0045] In another preferred embodiment, the objects include livestock (such as pigs, cattle, sheep, etc.).

[0046] In another preferred embodiment, the object is a rodent.

[0047] In another preferred embodiment, the object includes a rat or a mouse.

[0048] In another preferred embodiment, the data is derived from a sample of the brain tissue.

[0049] In another preferred embodiment, the sample includes slices, homogenates, or combinations thereof.

[0050] In a second aspect of the invention, an apparatus is provided for assessing the function of endogenous NMDA receptors in brain tissue selected from the group consisting of the cortex, hippocampus, or combinations thereof.

[0051] The device includes:

[0052] (M1) Input module, configured to: input data on the structure and / or quantity of endogenous NMDA receptors (NMDARs) in the brain tissue of a given subject, wherein the NMDA receptors include GluN1-N2A-N2B triheteromers; and

[0053] (M2) Analysis module, configured to: compare the structure and / or quantity of the GluN1-N2A-N2B triheteromeric molecule with a reference structure and / or quantity, thereby providing an evaluation of the function of endogenous NMDA receptors in brain tissue; and

[0054] (M3) Output module, which is configured to output the evaluation results.

[0055] In another preferred embodiment, the structural data is obtained by resolving the structure of the endogenous NMDA receptor (NMDAR) subunit.

[0056] In another preferred embodiment, the parsing method includes the steps of:

[0057] (Sa) Prepare specific antibodies against the GluN1, GluN2 and GluN3 subunits of the NMDA receptor;

[0058] (Sb) Separation and extraction of total protein from endogenous tissues;

[0059] (Sc) The specific antibody was mixed with total protein to separate and purify the subunit-antibody complex; and

[0060] (Sd) The structure of the complex was determined.

[0061] In another preferred embodiment, in step Sa, a recombinant NMDA receptor protein is used as an immunogen to generate specific antibodies against the GluN1, GluN2 and GluN3 subunits.

[0062] In another preferred embodiment, in step Sb, the endogenous tissue is selected from the group consisting of the cerebral cortex and the hippocampus.

[0063] In another preferred embodiment, in step Sd, the subunit-antibody complex is structurally resolved using cryo-electron microscopy.

[0064] In another preferred embodiment, the NMDAR is a heterotetramer composed of two fixed GluN1 subunits and two variable GluN2 / 3 subunits.

[0065] In another preferred embodiment, the GluN2 includes GluN2A, GluN2B, GluN2C, and GluN2D.

[0066] In another preferred embodiment, the GluN3 includes GluN3A and GluN3B.

[0067] In another preferred embodiment, the subunit composition types of the NMDAR include: GluN1-N2A-N2B triisotetramer, GluN1-N2A and GluN1-N2B diisotetramer.

[0068] In another preferred embodiment, the proportions of GluN1-N2A-N2B, GluN1-N2A, and GluN1-N2B in NMDAR are 45%, 35%, and 20%, respectively.

[0069] In a third aspect of the invention, a method for screening drugs is provided, the method comprising the steps of:

[0070] (S1) In the experimental group, the test compound was administered to the subjects, and data on the structure and / or quantity of endogenous NMDA receptors (NMDAR) in the brain tissue of the subjects were obtained DT1; in the blank control group, the test compound was not administered to the subjects and other experimental conditions were the same, and data on the structure and / or quantity of endogenous NMDA receptors (NMDAR) in the brain tissue of the subjects were obtained DT2.

[0071] The NMDA receptor includes the GluN1-N2A-N2B triisotetramer, and the brain tissue is selected from the group consisting of the cortex, hippocampus, or combinations thereof; and

[0072] (S2) Compare the data DT1 with the data DT2.

[0073] Specifically, if the quantitative characteristics of GluN1-N2A-N2B triheteromers in the brain tissue of the experimental group are closer to the quantitative reference value compared with the blank control group, it suggests that the tested compound is a candidate drug for improving brain function; and / or

[0074] In particular, if the structural characteristics of the GluN1-N2A-N2B triheteromer in the brain tissue of the experimental group are closer to the reference structure than those of the blank control group, it suggests that the test compound is a candidate drug for improving brain function.

[0075] In another preferred embodiment, in step (S2), if the number of GluN1-N2A-N2B triisotetramers in the brain tissue of the experimental group is significantly higher than that in the brain tissue of the blank control group compared with the blank control group, then the test compound is suggested to be a candidate drug for improving brain function.

[0076] In another preferred embodiment, the brain function is selected from the group consisting of cognitive function, mental state, memory function, or a combination thereof.

[0077] In another preferred embodiment, in step (S2), a comparison of structural features selected from the following group is performed:

[0078] (i) Compare the centroid distance (COM) (preferably, compare the COM distance between GluN1-R1 and GluN2-R1, between GluN2-R1 and GluN2-R2, and / or the rotation angle between the two leaflets of GluN2-R2);

[0079] (ii) Compare the structural changes of the GluN1-N2A-N2B triheteromer in the presence of NMDA receptor allosteric modulators;

[0080] (iii) Compare the structural features of the LBD intradimer interface and the LBD interdimer interface;

[0081] (iv) Compare the conformational changes of the GluN2B subunit between GluN1-N2B and GluN1-N2A-N2B;

[0082] (v) Compare the conformational changes of the GluN2A subunit between GluN1-N2A and GluN1-N2A-N2B.

[0083] In another preferred embodiment, the experimental subject is a non-human mammal.

[0084] In another preferred embodiment, the experimental subject is a non-human mammalian model in which the relative levels of GluN1-N2A-N2B in the brain tissue are significantly lower than the reference levels.

[0085] In another preferred embodiment, the reference content is 45% GluN1-N2A-N2B.

[0086] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0087] Figure 1 The purification, biochemical analysis, and cryo-electron microscopy results of eNMDARs are shown:

[0088] (A) Flowchart of affinity chromatography for the purification of eNMDARs from the cerebral cortex and hippocampus of adult rats. Twin-StrepII labeling on Fab 4F11 The C-terminus of the heavy chain is shown in red;

[0089] WB analysis of the solubilized (Solu) and flow-through (Ft) samples during the purification process in (B)(A);

[0090] (C) SEC map of isolated endogenous proteins; this map shows the Western blots (WB) of the GluN1, GluN2A, and GluN2B subunits for each corresponding SEC score. The red boxes highlight the scores used for subsequent biochemical and cryo-electron microscopy analyses.

[0091] (D) The pie chart is a semi-quantitative mass spectrum of the eNMDAR subunit;

[0092] (E) Cyro-EM structures of endogenous macromolecular complexes. The top image shows representative 2D class averages, and the bottom image shows cryo-electron microscopy. Scale bar.

[0093] Figure 2 This shows three major eNMDAR subtypes in the adult cerebral cortex and hippocampus:

[0094] (AC) Cryo-electron microscopy images of GluN1-N2A-B2B trisubunit eNMDAR (A), GluN1-N2B disubunit eNMDAR (B), and GluN1-B2A disubunit eNMDAR (C). GluN1, GluN2A, and GluN2B subunits are indicated in gray, orange, and purple, respectively; GluN1-Fab 4F11 GluN2A-Fab 28CGluN2B-Fab2, n-chain polysaccharide, and antagonist (R)-CPP are represented by blue, mint, pink, purple, and gold, respectively. The overall structure of eNMDAR consists of three layers: the n-terminal domain (NTD), the ligand-binding domain (LBD), and the transmembrane domain (TMD), shown in a side view (top) and a top-down view (bottom). In (C) (top), the bilobed shells of NTD and LBD are labeled as R1 / R2 and D1 / D2 lobes, respectively. The 2D class averages in (A and B) show representative top-down views, with Fab density highlighted by colored arrows (scale bar). );

[0095] (D) The violin plot of scRNA-seq analysis shows the mRNA levels of seven NMDAR genes in excitatory / inhibitory neurons and non-neuronal cells in the cortex and hippocampus of adult mice.

[0096] Figure 3 The asymmetric structure of GluN1-N2A-N2B is shown:

[0097] (A) Atomic model of GluN1-N2A-N2B tri-eNMDAR. The subunits GluN1 (A chain), GluN2A, GluN1 (C chain), and GluN2B are gray, orange, light green, and purple, respectively; the right figure shows a top-down view of the NTD and LBD layers, illustrating the centroid (COM) distance between two NTDs or LBDs. Cartoon diagrams illustrate dimer formation within NTD or LBD layers and domain exchange characteristics between the two layers. In the LBD layer, the intradimer interface and interdimer interface of the LBD dimer are marked with arrows;

[0098] (B) Cartoon diagrams of GluN1(A)-GluN1(C) (left) and GluN2A-GluN2B (right). In the left diagram, GluN1(C) is aligned with GluN1(A), LBDs are superimposed, and the rotation angle between NTD and TMD is marked. The right diagram shows the NTD-LBD vector angle formed by the connection of the D1 and D2 lobes of the GluN2 subunits with the COMs of R2 and R1;

[0099] (C) Structural comparison of two NTD (top) or two LBD (bottom) heterodimers. The NTD or LBD heterodimers are arranged with the GluN2-R1 or GluN2-D1 lobes, respectively. The top figure shows the COM distances between GluN1-R1 and GluN2-R1, GluN2-R1 and GluN2-R2, and the rotation angle between the two GluN2-R2 lobes. The bottom figure shows the COM distances between the GluN1-D1 and GluN2-D1 lobes;

[0100] (D) Cartoon diagram of the NTD-LBD transition observed at the LBD intermediate dimer interface of the GluN2A (left) and GluN2B (right) subunits. Colored circles represent the COM of the R2 lobes, D1 lobes, and αE helices of the GluN1 or GluN2 subunits, and indicate the distances between them;

[0101] (E) Top-down TMD architecture diagram, as shown in the figure. This represents the COM distance between two adjacent M3 threaded screws;

[0102] (F) The left panel shows the HOLE analysis of tri-eNMDAR. The middle and right panels are side views of the TMD arrangements of GluN1(A)-N2A and GluN1(C)-N2B in opposite directions, marked with eye tags in (E). The comparison of the Cα-Cα distances of the four residue pairs between GluN2A / N2B and adjacent GluN1 subunits indicates that the GluN2-M2 / M3 and GluN1-M3 arrangements are asymmetrical.

[0103] Figure 4 The conformational differences between GluN1-N2B di-eNMDARs and GluN1-N2A-N2B tri-eNMDARs are shown:

[0104] (A) Atomic model of GluN1-N2B. The middle figure shows a top-down view of the NTD and LBD layers, showing the COM distance between the GluN1 and GluN2B NTD or LBD. The right figure compares the NTD tetramer interface of GluN1-N2B and GluN1-N2A-N2B formed by two GluN2-R2 lobes, showing the COM distance between the two α5 helices or GluN2-R2 lobes;

[0105] (B) Structural comparison of GluN2BNTD (left) or LBD (right) heterodimers between GluN1-N2B (color-coded) and GluN1-N2A-N2B (transparent gray) heterodimers. The leaflet arrangements of the NTD or LBD heterodimers overlapping with GluN2-R1 or GluN2-D1, respectively. The COM distances between the GluN1-R1 and GluN2B-R1, GluN2B-R1 and GluN2B-R2, and GluN1-D1 and GluN2B-D1 leaflets are marked. The rotation angle of the GluN2B-R2 leaflet from di- to tri-eNMDAR is shown in the figure. The red box shows the GluN2B-specific efendiol binding site;

[0106] (C) Comparison of the GluN1-GluN2B NTD heterodimer interface of GluN1-N2B with GluN1-N2A-N2B (top) or effendil-bound recombinant structures (bottom). The model is superimposed on the GluN2B-R1 leaflet, showing the relative rotation angle of the GluN2B-R1 leaflet from di-eNMDAR to tri-eNMDAR or effendil-bound state. The change in the COM distance between GluN1-α3 and GluN2B-α2 indicates the binding of effendil;

[0107] (D) TMD comparison of GluN1-N2B di-eNMDAR and GluN1-N2A-N2B tri-eNMDAR. The model is aligned with the M3 helix of the four subunits. COM distances are indicated between the M3 helices of the di- (black letters) and tri- (gray letters) eNMDARs. Light green arrows highlight the relative displacement of GluN1(C)-M3 from the symmetric di-eNMDAR to the asymmetric GluN1-N2A-N2B tri-eNMDAR;

[0108] (E) Schematic diagram of conformational changes of the GluN2B subunit between GluN1-N2B and GluN1-N2A-N2B. Subunits are indicated by the corresponding colors in the previous diagram. Arrows highlight: the detwisting of GluN2B-NTD, the formation of a tighter GluN1-N2B NTD and LBD heterodimer through the R1-R1 and D1-D1 interfaces, and the approach of GluN1(C)-M3 to GluN2B-M3.

[0109] Figure 5 Antibody identification and isolation of eNMDARs in the brain are shown:

[0110] (A) Under confocal immunofluorescence microscopy, HEK293T cells expressing GFP were fused with GluN1 (with or without the n-terminal alternatively spliced ​​exon 5 motif) and GluN2 or GluN3 subunits, and were respectively analyzed with mAb.4F11 Fab2, mAb 28C mAb 5D5 or mAb 4D5 Staining. Combine the fluorescence signals of GFP, DAPI (blue), and IgG (red). Scale bar, 10 μm;

[0111] (B) SEC maps (black map) of recombinant GluN1-N2A, GluN1-N2B, GluN1-N2C, and GluN1-N2D receptors, and Fab 4F11 (Blue image) and the corresponding GluN2-specific fab. The fab was mixed with NMDAR at a molar ratio of 1:5. A magnified view of the normalized peaks highlights the changes in elution volume.

[0112] (C) Binding mode of GluN2-specific fabs. 2D projections of the recombinant GluN1-N2A (EMDB-9158), GluN1-N2A-N2C (EMDB-33791), and GluN1-N2D (EMDB-33792) receptors without fab binding were obtained by low-pass screening. The spectra were generated. Representative two-dimensional class averages of GluN2 fab-bound recombinant NMDARs were processed using 120 kV cryo-electron microscopy. Arrows indicate additional Fab density. Scale bar, 10 nm.

[0113] (D) Confocal immunofluorescence microscopy of HEK293T cells expressing chimeric GluN1-N2 receptors, in which NTD, S1, and S2 fragments are exchanged between GluN2A and GluN2D subunits, and GluN2A-fab is used. 28c Staining. The NTD, S1, and S2 of rat GluN2A are composed of residues L34-W390, H405-S556, and Q661-Q811, respectively. The S1 of rat GluN2D contains residues H428-A581.

[0114] (E) Fab using double-stranded tags 4F11 Workflow for purifying whole-pig brain eNMDAR.

[0115] (F) SEC chromatograms of eNMDARs purified, with an inset showing each SEC fragment targeting the GluN1 subunit and Fab. 4F11 Western blot analysis of Strep tags on heavy chains. Collect GluN1 and Fab shown in the red box. 4F11 The two portions with the highest concentrations were examined under negative staining electron microscopy.

[0116] SDS-PAGE silver staining analysis of each SEC portion in (G) and (F). The comparison of the eluted sample with the two negatively stained portions is shown in the figure.

[0117] (H) Typical negative stained EM image and eNMDAR two-dimensional average. Arrows indicate Fab. 4F11 Density. Scale bar, 10 nm.

[0118] Figure 6 MS and XL-MS analyses of eNMDAR and co-purified proteins in the rat cerebral cortex and hippocampus are shown:

[0119] (A) Figure 1 Silver-stained SDS-PAGE analysis of each SEC fragment corresponding to C. The mixing fraction used for cryo-electron microscopy sample preparation is indicated in red. The gel was cut into 5 bands (bands 1 to 5) for enzymatic digestion and mass spectrometry analysis, as shown in the green boxes. The middle figure shows a summary table of the protein intensities of the top 6 integrative peptides in each band (sorted from highest to lowest intensity). The pie chart on the right shows the composition of the protein complexes with the highest intensity in the cryo-electron microscopy sample.

[0120] (B) Identification and semi-quantification of eNMDAR subunits in full-band integrated mass spectrometry data. Peptide profile matching (PSM) and intensity are averages of two repeated injection mass spectrometry samples.

[0121] (C) The top figure shows a schematic diagram of eight GluN1 splicing isomers, illustrating the motif sequence encoded by four exons through the insertion of exons 5 and 21, and the insertion of exons 22 or 22'. The bottom figure shows the unique peptides of the GluN1 selective splicing motif detected in cryo-electron microscopy samples.

[0122] (D) CTD cleavage of GluN2A and GluN2B subunits. Scissor marks on the CTD represent potential enzyme cleavage sites. Full-length (~200 kDa), CTD cleavage (~120 kDa), and CTD fragments (10–40 kDa) of the GluN2A / N2B subunits are present in bands 1, 2, and 5, respectively. The bar chart shows the intensity differences of the NTD peptide (shared by both full-length and CTD cleavage forms) and the CTD peptide (shared by both full-length and CTD fragment forms) in bands 1, 2, and 5.

[0123] (E) The left figure shows the crosslinking network identified between eNMDAR and the co-purified protein, with a false discovery rate (FDR) ≤ 1% (black line). The crosslinks were obtained from the XL-MS database of adult mouse hippocampal synapses (red line). The right table lists the identified crosslinked lysine pairs between the complex subunits shown in the network, with the crosslinked lysine residues highlighted in red.

[0124] Figure 7 The eNMDAR Cryo-EM data processing flow is shown:

[0125] (A) Iterative "Build and Retrieve" (BaR) process for purifying endogenous proteins from the cerebral cortex and hippocampus of adult rats. A total of 9190k heterogeneous particles were classified in two dimensions to remove ice and carbon film background particles. The cleaned 3932k particle pile was then classified according to structural differences, and an initial model was iteratively built to retrieve particles from the pile. Maps of six different endogenous complexes, including eNMDARs, were generated.

[0126] (B) Cryo-EM data processing of the eNMDAR-Fab complex. Two-dimensional class means with blurred extracellular regions are highlighted with red dashed boxes. After focused 3D classification and heterogeneous refinement, the data are shown in blue (Fab). 4F11 ), mint (Fab 28C The arrows marked with pink (Fab2) indicate the three different Fab densities. Fourier shell correlation (FSC), local resolution estimates, and angular distributions of the final refined map are presented at the gold standard of 0.143.

[0127] Figure 8 The structural analysis of GluN1-N2A-N2B tri-eNMDAR is shown:

[0128] (A and B)GluN1 Fab 4F11 (A) and GluN2A Fab 28C (B) Antigen-antibody interaction analysis. GluN1-NTD and Fab 4F11 Combined (heavy chain is dark blue, light chain is light blue) and GluN2A-LBD with Fab 28C The local density and structure diagrams of the binding (heavy chains are dark green, light chains are light red) are shown in the figure. A magnified view of the interface between the epitope and the complementarity-determining region (CDR) of the Fab heavy and light chain epitopes is shown at the bottom. Key residues and the n-chain glycans mediating the interaction are marked, and their densities are represented by a gray grid.

[0129] (C) Comparison of the conformations of GluN2A-NTD in GluN2A-N2B tri-eNMDAR with recombinant GluN2A-NTD that is unbound (left; PDB 6MMP) or bound (right; PDB 5TPW). Models from natural and recombinant NMDARs are shown in orange and transparent gray, respectively. The models are superimposed on the R1 lobe to show the relative rotation between the R2 lobes and the COM distance between the R1 and R2 lobes.

[0130] (D) The left panel shows a conformational comparison of (R)-CPP binding to GluN2-LBDs in tri-eNMDAR with that of glutamate-binding GluN2-LBDs in recombinant GluN1-N2A (PDB6MMP) and GluN1-N2B (PDB7TE9) receptors. The model is superimposed on the D1 lobe to obtain the relative rotation between the D2 lobes and the COM distance between the R1 and R2 lobes. The right panel shows the EM density and structural coordinates of (R)-CPP on the LBD shells of the GluN2A and GluN2B subunits in tri-eNMDAR.

[0131] In each figure, heterodimer is a heterodimer, and interface is an interface. Detailed Implementation

[0132] Through extensive and in-depth research, the inventors, for the first time, used heterologously expressed NMDA receptors to immunize mice, producing and screening specific monoclonal antibodies targeting each subunit of the NMDA receptor. They successfully enriched and isolated endogenous NMDA receptors from the cerebral cortex and hippocampus of adult rats, and further elucidated the subunit composition, assembly, and structure of the NMDA receptor using cryo-electron microscopy. Based on these findings, this invention was completed.

[0133] the term

[0134] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0135] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0136] In this invention, the terms "NMDA receptor" and "NMDAR" are used interchangeably, both referring to the N-methyl-D-aspartate receptor.

[0137] In this invention, the term "eNMDAR" refers to the endogenous N-methyl-D-aspartate receptor.

[0138] In this invention, the terms "di-subunit eNMDAR", "di-iso-eNMDAR" and "di-eNMDAR" are used interchangeably and all refer to the tetrameric form of NMDA receptors having two different subunits.

[0139] In this invention, the terms "tri-subunit eNMDAR", "triiso-eNMDAR" and "tri-eNMDAR" are used interchangeably and all refer to the tetrameric form of an NMDA receptor having three different subunits.

[0140] NMDA receptor

[0141] The NMDA receptor (N-methyl-D-aspartic acid receptor) is a subtype of ionotropic glutamate receptors that is permeable to potassium. + Na + Ca2 + With its complex molecular structure and unique pharmacological properties, the NMDA receptor plays a crucial physiological role in the development of the nervous system, such as regulating neuronal survival, the development of dendrites and axons, and participating in the formation of synaptic plasticity. In mammals, the NMDA receptor is encoded by seven different genes, forming seven different subunits. Functional NMDA receptors typically assemble from two essential GluN1 subunits and two variable GluN2 / 3 (N2A-N2D, N3A-N3B) subunits, forming a diiso- or triiso-tetramer.

[0142] In this invention, three main subtypes of the NMDA receptor were determined: GluN1-N2A-B2B trisubunit eNMDAR, GluN1-N2B, and GluN1-B2A disubunit eNMDAR, accounting for approximately 45%, 35%, and 20% of the NMDA receptor, respectively. Furthermore, the tri-eNMDAR tetramer and the di-eNMDAR tetramer have different conformations, and the same subunit also exhibits different conformations in the tri-eNMDAR and di-eNMDAR subtypes.

[0143] Methods for assessing endogenous NMDA receptor function

[0144] The present invention also provides a method for assessing the function of endogenous NMDA receptors in brain tissue, the method comprising the steps of:

[0145] (a) Obtaining data on the structure and / or quantity of endogenous NMDA receptors (NMDARs) in the brain tissue of a subject; and

[0146] (b) The structure and / or quantity of the NMDA receptors are compared with reference structures and / or quantity values ​​to provide an evaluation of the function of endogenous NMDA receptors in brain tissue.

[0147] Specifically, this invention determined reference structures for three major NMDA receptor subtypes, including the GluN1-N2A-N2B triisotetramer, the GluN1-N2A diisotetramer, and the GluN1-N2B diisotetramer, as shown in the accompanying drawings, and provides more specific structural data, for example,

[0148] (1) The COM distance between GluN1-R1 and GluN2-R1, between GluN2-R1 and GluN2-R2, and the rotation angle between the two leaflets of GluN2-R2;

[0149] (2) Structural changes of GluN1-N2A-N2B triheteromer in the presence of NMDA receptor allosteric modulators;

[0150] (3) Structural features of the LBD intradimer interface and the LBD interdimer interface in GluN1-N2A-N2B;

[0151] (4) Conformational changes of the GluN2B subunit between GluN1-N2B and GluN1-N2A-N2B.

[0152] In addition, the present invention provides reference contents for three major NMDA receptor subtypes, namely GluN1-N2A-N2B, GluN1-N2A and GluN1-N2B, accounting for 45%, 35% and 20% of total NMDAR, respectively.

[0153] Therefore, the NMDA receptors in the sample can be functionally evaluated based on the reference structure and reference content of the aforementioned NMDA receptor subtypes.

[0154] Evaluation equipment

[0155] The present invention also provides an apparatus for assessing the function of endogenous NMDA receptors in brain tissue, the apparatus comprising:

[0156] (M1) Input module, the input module being configured to: input data on the structure and / or quantity of endogenous NMDA receptors (NMDARs) in the brain tissue of a given subject; and

[0157] (M2) Analysis module, configured to: compare the structure and / or quantity of the NMDA receptor with reference structural and / or quantity reference values, thereby providing an evaluation result of the function of endogenous NMDA receptors in brain tissue; and

[0158] (M3) Output module, which is configured to output the evaluation results.

[0159] More specifically, the present invention also provides a method for resolving the structure of endogenous NMDA receptor subunits, comprising the following steps:

[0160] (Sa) Using recombinantly expressed NMDA receptor protein as an immunogen, specific antibodies against the GluN1, GluN2 and GluN3 subunits of the NMDA receptor were prepared.

[0161] (Sb) Separation and extraction of total protein from endogenous tissues;

[0162] (Sc) The specific antibody was mixed with total protein to separate and purify the subunit-antibody complex; and

[0163] (Sd) The structure of the complex was determined.

[0164] Among them, GluN2 includes four types: GluN2A, GluN2B, GluN2C and GluN2D, while GluN3 includes two types: GluN3A and GluN3B.

[0165] Drug screening methods

[0166] The present invention also provides a method for screening drugs, the method comprising the steps of:

[0167] (S1) In the experimental group, the test compound was administered to the subjects, and data on the structure and / or quantity of endogenous NMDA receptors (NMDARs) in the brain tissue of the subjects were obtained (DT1); in the blank control group, the test compound was not administered to the subjects, and all other experimental conditions were the same, and data on the structure and / or quantity of endogenous NMDA receptors (NMDARs) in the brain tissue of the subjects were obtained (DT2); and

[0168] (S2) Compare the data DT1 with the data DT2.

[0169] Specifically, if the quantitative characteristics of GluN1-N2A-N2B triheteromers in the brain tissue of the experimental group are closer to the quantitative reference value compared with the blank control group, it suggests that the tested compound is a candidate drug for improving brain function; and / or

[0170] In particular, if the structural characteristics of the GluN1-N2A-N2B triheteromer in the brain tissue of the experimental group are closer to the reference structure than those of the blank control group, it suggests that the test compound is a candidate drug for improving brain function.

[0171] Under the guidance of this invention, a model of NMDA receptor deficiency can be constructed as an experimental subject. Compared with the blank control group, if the structure and number of NMDA receptor subtypes in the experimental group are closer to the reference standard given in this invention after the administration of the candidate drug, it indicates that the drug has the potential to improve NMDA receptor function.

[0172] Compared with the prior art, the advantages of the present invention are as follows:

[0173] 1. This invention has identified three major receptor subtypes of NMDA: GluN1-N2A-N2B triisotetramer, GluN1-N2B and GluN1-N2A diisotetramer, and these three subtypes constitute NMDA receptors in the cortex and hippocampus at proportions of approximately 45%, 35% and 20%, respectively.

[0174] 2. This invention discovers that the GluN1-N2A-N2B triisotetramer is the most abundant endogenous subtype (comprising 45% of the NMDA receptor), updating the concept in the field that the GluN1-N2A and GluN1-N2B diisotetramers are the main subtypes for the past thirty years, and emphasizing the functional integration of the GluN2A and GluN2B subunits in physiological states.

[0175] 3. This invention discovers conformational changes of the GluN2B subunit in the GluN1-N2A-N2B triisotetramer and GluN1-N2B diisotetramer, revealing the phenomenon of conformational differences of the same subunit in different receptors. This provides the possibility for designing compounds that not only have subunit specificity but also receptor subtype preference, which helps to improve the targeting and specificity of NMDA receptor drugs.

[0176] 4. This invention reveals the assembly and structure of endogenous NMDA receptors, laying the foundation for subsequent research on the pathological changes in NMDA receptor assembly and structure under brain disease states, and revealing the in vivo mechanism of action of clinical drugs targeting NMDA receptors.

[0177] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0178] Experimental materials

[0179] Insect Sf9 cells (female, ATCC CRL-1711).

[0180] High Five cells (female, Thermo Fisher Scientific BTI-TN-5B1-4).

[0181] HEK 293T cells (female, ATCC CRL-3216) were maintained in a 5% (v / v) CO2 incubator with DMEM supplemented with 10% fetal bovine serum at 30 or 37°C.

[0182] Wild-type adult (8-week-old) male Wistar rats were purchased from the Charles River Laboratory in China. The prefrontal cortex and hippocampus of the rats were dissected without feeding, resting, or other experimental procedures.

[0183] Brain tissue from a wild-type boar (approximately 2 months old) was obtained from the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

[0184] Experimental methods

[0185] Preparation and purification of monoclonal antibodies against NMDAR subunits

[0186] To generate monoclonal antibodies against different NMDAR subunits, GluN1-N2A receptor, 16 GluN1-N2C and GluN1-N2D receptors were expressed and purified as immunogens to generate antibodies against GluN1, GluN2A, GluN2C and GluN2D subunits.

[0187] Recombinant NMDAR protein was purified in Tris-buffered saline (TBS: 150 mM NaCl, 20 mM Tris, pH 8.0) containing 0.1% digitoxin, and then mixed with Freund's complete adjuvant at a 1:1 (v / v) ratio. BALB / c mice were intraperitoneally injected with 100 μg of the emulsified protein and immunized every two weeks.

[0188] After the third injection, spleen cells were collected and fused with SP2 / 0 cells using PEG1450 to generate hybridomas. Positive clones were screened using a live cell method, and monoclonal antibodies of GluN1, GluN2A, GluN2C, and GluN2D produced by the 4F11, 28C, 5D5, and 4D5 hybridoma cell lines were identified.

[0189] GluN1 Fab 4F11GluN2B Fab2 was generated using the Bac-to-Bac TOPO expression system (Invitrogen, A11339). DNA sequences of the variable fragment (Fv) regions of the heavy and light chains of Fab4F11 and Fab2 were cloned from hybridoma cell mRNA and synthesized separately by PCR amplification. The Fv region DNA sequences were inserted into a bicisic pFastBac vector containing a humanized Fab constant fragment. The GP64 signal peptide (MVSAIVLYVLLAAAAHSAFA) was added to the N-terminus of the heavy and light chains, respectively. Twin-Strep Tag Il and 8xHis tags were added to the C-terminus of the Fab4F1 heavy and light chains, respectively, and Twin-Strep Tag Il was added to the C-terminus of the Fab2 light chain. Baculoviruses encoding the constructed Fab4F11 and Fab2 were prepared at a concentration of 2 × 10⁻⁶. 6 High Five cells were infected at a density of / ml at 27°C. 96 hours after infection, the pH of the culture supernatant was adjusted to 8.0, and the supernatant was collected after centrifugation at 7000g for 30 minutes. Twin-Strep labeled FabaF11 and Fab2 were purified by affinity chromatography, concentrated in phosphate-buffered saline (PBS, pH 7.4), and dialyzed.

[0190] The monoclonal antibody was digested with papain to obtain GluN2A-Fab. 28c GluN2C-Fab 5D5 and GluN2D-Fab 4D5 (Papain and monoclonal antibody digestion ratio: 1:20, w / w). Hybridoma cells were intraperitoneally injected into mice to generate ascites. The monoclonal antibody was purified from the ascites using rProtein-G Sepharose (smart-lifessciences, SA016025). After papain digestion, the fabs were purified by anion exchange chromatography (wash buffer: 20 mM Tris-HCl, pH 10.8; elution buffer: 20 mM Tris-HCl, pH 10.8, 1 M NaC). The purified fabs were then stored in PBS.

[0191] Live cell immunostaining

[0192] HEK 293T cells were transiently transfected with plasmids encoding GluN1-N2A, GluN1-N2B, GluN1-N2C, and GluN1-N2D (plasmid to polyethyleneimine ratio 1:3, w / w). After 48 h, cells on coverslips were gently washed with PBS and then incubated with 60 μg / ml monoclonal antibody for 30 min. Cells were washed with PBS, fixed with 4% p-hydroxyformaldehyde for 5 min, and then incubated with secondary antibody (Invitrogen, a-21089) at a 1:1000 dilution for 20 min. After washing, coverslips were mounted with DAPI-containing extended gold anti-fading reagent (Thermo Fisher, 62248) and imaged using an Olympus FV3000 confocal microscope.

[0193] Isolation of NMDA receptors from brain tissue

[0194] Protein purification was performed using 8-week-old male Wistar rats. Brains from 80 rats were collected, washed with cold water TBS buffer, and the cerebral cortex and hippocampus were separated. The brain tissue from these two regions was rapidly frozen in liquid nitrogen and stored at 80°C. For preliminary detergent screening, antibody detection, and purification of the natural receptor, pig brains were used to ensure sufficient source and reduce animal numbers. The frozen tissue was homogenized and sonicated in 120 ml of low-temperature TBS buffer containing 1.6 μM aprotinin, 4 mM gasstatin A, 4 μg trypsin mr, and 2 mM benzoyl fluoride (PMSF). The homogenate was centrifuged at 10,000 g for 15 min to remove cell debris. The supernatant was centrifuged at 200,000 g for 1 h at 4°C, and the membrane fraction was collected. The membrane fraction was resuspended and dissolved in TBS buffer with 2% dodecyl maltose neopentyl glycol (L-MNG). 0.2% steranoic acid cholesterol (CHS) and protease inhibitors were mixed at 4°C for 3 h. After 1 hour of ultracentrifugation at 200,000 g, 2 mg of Twin-Strep-labeled GluN1Fab4F11 and 5 ml of Biolock solution (IBA, 2-0205-250) were added to the supernatant and incubated for 1.5 hours, followed by incubation with one streptactin resin for 1 hour. Finally, the resin was collected by gravity flow column, washed with washing buffer (TBS supplemented with D.1% L-MNG, 0.01% CHS, and 100 pM EDTA), and eluted with washing buffer supplemented with 10 mM d-dethiobiotin. The eluted protein was then reacted with excess GluN2A-Fab4F11. 28c GluN2B-Fab2, GluN2C-Fab 5D5 and GluN2D-Fab 4D5Incubate for 1 hour at 0.1 mg / ml for each concentration. Afterward, concentrate the mixture and further purify it using a Superose 6 10 / 300GL column (GE Healthcare) in TBS buffer supplemented with 0.1% digitalisin, 5 μM CHS, 0.1 M CHAPSO, and 100 μM EDTA.

[0195] Negative EM

[0196] The purified whole-pig brain protein was diluted to a concentration of 0.03 mg ml⁻¹. A 3 μl sample was placed on a 400-mesh luminescent continuous carbon grid (Electron Microscopy China, Inc.) for 40 seconds. Excess sample was then removed with a small piece of filter paper, and the sample was immediately washed twice with deionized water and incubated in 2% fresh uranyl acetate solution for 40 seconds. Excess uranyl acetate solution was then further removed with filter paper, and the sample was air-dried at room temperature and examined using a 120 kV Talos L120C electron microscope (FEI) at a magnification of 92,000x, corresponding to a pixel size of [missing information]. Total dose set to

[0197] Cryo-electron microscopy sample preparation and data acquisition

[0198] The purified rat cerebral cortex and hippocampal natural proteins were diluted to 0.035 mg / ml. -1 The concentration of the protein sample was determined, and the competitive antagonist (R)-CPP (TOCRIS, 0247-10) was added to a final concentration of 1 mM before grid preparation. 3 μL of protein sample was dropped onto a luminescent 300-mesh quantitative foil R1.2 / 1.3 (Cu) grid, which was covered with a 2 nm continuous carbon film. The grid was imprinted at 8 °C and 100% humidity for 4 s, and then immediately immersed in liquid ethane using a FEI Vitrobot (Thermo Fisher).

[0199] Cryo-electron microscopy data were acquired on a 300 kV Titan Krios G3 electron microscope (FEI) equipped with a K3Summit direct electron detector (Gatan) and a GIF quantum energy filter. The video magnification is 81,000x, and the corresponding pixel size in super-resolution mode is [missing information]. Typical defocus values ​​range from -1.0 to -2.5 μm. The film stack is dose-graded over 50 frames, with a total dose of... Automatic data acquisition was performed using a series EM 3.7.11.

[0200] Example 1. Isolation of endogenous NMDA receptors in the brain

[0201] The subunits of the NMDA receptor and their possible assembly types, such as Figure 1 As shown.

[0202] Mice were immunized with purified recombinant GluN1-N2 receptor, resulting in four monoclonal antibodies: mAb4F11, mAb28C, mAb5D5, and mAb4D5, which recognize the GluN1, GluN2A, GluN2C, and GluN2D subunits, respectively. Furthermore, the GluN2B-specific antigen-binding fragment, Fab2, was cloned and recombinantly expressed. Live-cell immunostaining and size exclusion chromatography (SEC) analyses showed that each antibody specifically binds to the tetramer-formed NMDAR (Number Methyl Methyl Adhesive Receptor). Figure 5 Cryo-electron microscopy analysis of Fab-bound recombinant NMDARs showed that GluN2A-fab 28c GluN2C-fab 5d5 and GluN2D-fab 4d5 They bind to the GluN2A, GluN2C, and GluN2D subunits respectively in different binding configurations. Figure 5 C). In particular, this further confirms mAb 28C The H405-S556 fragment targeting the ligand-binding domain (LBD) of the GluN2A subunit ( Figure 5 D).

[0203] Next, the use of fab-based 4F11 The feasibility of affinity chromatography for capturing eNMDARs from porcine brain via the GluN1 subunit was investigated. Membrane fractions were collected by differential centrifugation and dissolved in maltose-neopentyl glycol (MNG) detergent. Fab was labeled with Twin-Strep II at the C-terminus of the heavy chain. 4F11 And added to the dissolved sample to capture eNMDARs ( Figure 5 E). Western blot analysis of each SEC fragment showed that the GluN1 subunit was widely distributed (E). Figure 5 F), silver staining showed that the purified native protein exhibited heterogeneity. Figure 5 G). The two components with the highest concentrations of GluN1 and Fab4F11 were collected and detected by negative staining electron microscopy. Two-dimensional (2D) class averages show a typical side view of NMDARs, with their outer portions bound to the two Fab densities (G). Figure 5 H). Therefore, a method for capturing tetrameric eNMDARs from brain tissue using fab4f11-based affinity chromatography was successfully established.

[0204] Example 2. Structural determination of endogenous proteins

[0205] Next, at 8 weeks, the cerebral cortex and hippocampus of adult rats were dissected to establish a purification pipeline. Figure 1 A). Western blot analysis showed that most of the dissolved eNMDARs were absorbed by Fab. 4F11 Effective capture ( Figure 1 B). Then, four GluN2-fab-labeled affinity chromatography molecules were added, followed by elution of candidate GluN2 subunits from the sample. Finally, SEC was performed, and the fraction with a wide distribution of GluN1 subunits was collected for further biochemical and cryo-electron microscopy analysis. Figure 1 C). Mass spectrometry (MS) analysis showed that the eNMDAR subunit, 2-oxogluconate dehydrogenase complex (OGDHC), glutamine synthase (GS), ATP synthase, vacuolar adenosine triphosphatase (V-ATPase), calcium / calmodulin-dependent protein kinase II (CaMKII), and spectrin were most abundant in the purified sample. Figure 6 A). Among them, CaMKII and spectrin have been reported to interact directly with the C-terminal domain (CTD) of eNMDARs, regulating their transport, anchoring, and postsynaptic signal transduction.

[0206] Among eNMDARs, the GluN1 peptide showed the highest integration strength, followed by the GluN2B, GluN2A, and GluN3A subunits, which showed moderate integration strength. The GluN2D and GluN3B subunits showed the lowest integration strength. Figure 1 D and Figure 6 B). Furthermore, unique peptides encoding GluN1 candidate splicing motifs from exons 5, 21, 22, and 22′ were detected, indicating the presence of multiple GluN1 splicing isoforms in the purified eNMDARs. Figure 6 C). Western blot analysis using NTD antibodies showed that, in the solubilized samples, the molecular weights of the GluN2A and GluN2B subunits ranged from ~130 kDa to over 180 kDa. Figure 1 B), and in the final SEC component, the GluN2A and GluN2B subunits were mainly detected at ~130 kDa. Figure 1 C). MS data showed that approximately 90% of the CTDs of the GluN2A / B subunits were cleaved into fragments of 10-40 kDa. Figure 6 (D). This result indicates that GluN2-CTDs can be cleaved by endogenously calcium-activated neutral calpain.

[0207] The purified sample was treated with the GluN2 competitive antagonist (R)-CPP to stabilize eNMDARs in an inhibited state, and a cryo-electron microscopy dataset of 55,119 images was collected. Given the heterogeneity of proteins in the above biochemical analyses, a "build and retrieve" strategy was employed to separate particles belonging to macromolecular complexes with different characteristics in two-dimensional and three-dimensional classification. Figure 7 A). Reconstructed cryo-electron microscopy images of eNMDAR, GS, OGDHC, ATP synthase, V-ATPase, and respiratory complex I ( Figure 1 E), consistent with the protein with the highest intensity detected by mass spectrometry. To explore potential interactions between eNMDAR and other co-purified proteins, cross-linking mass spectrometry (XL-MS) was performed, and various cross-links between eNMDAR and ATP synthase, OGDHC, V-ATPase, or GS were identified. Figure 6 E). The most common cross-linking involves eNMDARs and ATP synthase. Functional studies have also suggested the presence of eNMDARs in the inner mitochondrial membrane, and the activation of mitochondrial eNMDARs enhances the activity of respiratory and ATP synthases.

[0208] Example 3. Cryo-electron microscopy structures of three major eNMDAR subtypes

[0209] Topaz training was further performed using particles of the eNMDAR-Fab complex, and particles were re-extracted from the original micrographs. In two-dimensional classification, Fab attachments to eNMDARs were found to correspond to different epitopes and orientations. Figure 7 B). After several rounds of global and 3D classification of GluN2-Fab, the three eNMDAR-fab complexes of GluN1-N2A-N2B tri-eNMDARs, GluN1-N2B and GluN1-N2A di-eNMDARs were respectively classified as follows: and The resolution is distinguished ( Figure 7 B). In the EM images of GluN1-N2A-N2B and GluN1-N2B eNMDARs, the local density of GluN2-LBDs and the antagonist (R)-CPP at the n-chain glycosylation site is clearly visible. Figure 2 (A and 2B).

[0210] Furthermore, based on the total particle size used in the final reconstruction, the proportions of GluN1-N2A-N2B triisoeNMDAR, GluN1-N2B, and GluN1-N2A diisoeNMDAR were 45%, 35%, and 20%, respectively. Figure 2 (A-2C). All eNMDARs consist of three tandem layers: an NTD, an LBD, and a transmembrane domain (TMD). The extracellular NTD (N-terminal domain) and LBD of each subunit are composed of bilobed, clamshell-like modules formed by R1 / R2 and D1 / D2 lobes, respectively. The three isotypes of eNMDARs assemble into a tetrameric arrangement, sharing the common characteristic of two essential GluN1 subunits in the peripheral position, characterized by two Fabs. 4F11Symmetrical binding of two GluN1-NTDs ( Figure 2 (A-2C). Among them, two Fabs 4F11 It also binds to both the R1 and R2 lobes of GluN1-NTD, with the heavy chain primarily interacting with the R1 lobe through hydrogen bonding, and the light chain primarily interacting with the R2 lobe through electrostatic interactions. Figure 8 A). Fab 4F11 The epitope is conserved among the N-terminal splice isomers of GluN1 and does not cross-interact with the adjacent GluN2 subunit. This binding mode ensures the separation of eNMDARs without bias towards isotypes or splice isomers.

[0211] The most abundant tri-eNMDARs subtypes are each represented by a Fab. 28C A Fab2 asymmetrically attached GluN2A-LBD and GluN2B-NTD were used for labeling. Figure 2 A). A Fab 28C Attached to the D1 leaf of GluN2A ( Figure 8 B), consistent with the epitope screening results of chimeric receptor live cell staining ( Figure 5 D). On the other hand, one Fab2 is bound to the R1 leaflet of GluN2B. Additionally, the two GluN1-N2B and GluN1-N2A diiso-eNMDAR isoforms are symmetrically labeled as either two Fab2s bound to GluN2B-NTD or two Fab2s bound to GluN2A-LBD. 28C ( Figure 2 B and 2C). Therefore, the main difference among all eNMDARs lies in the tetrameric arrangement, with two heterologous or homologous GluN2 subunits located near the central axis of the tetramer (B and C). Figure 2 A-2C). Furthermore, single-cell transcriptomic analysis of adult mice confirmed the high expression of GRIN1, GRIN2A, and GRIN2B in both neuronal and non-neuronal cell types. Figure 2 D). In summary, GluN1-N2A-N2B, GluN1-N2B, and GluN1-N2A are the three major subtypes in the adult cortex and hippocampus.

[0212] Example 4. Asymmetric structure of GluN1-N2A-N2B triiso-eNMDAR

[0213] GluN1-N2A-N2B tri-eNMDAR exhibits dimer arrangement and domain exchange characteristics between the NTD and LBD layers, allowing a given GluN1 promerogen to form different heterodimers with GluN2A and GluN2B subunits in the NTD and LBD layers. Figure 3A). To distinguish the two GluN1 protopolymers, they were labeled chain A and chain C, respectively. The global root mean square deviation (RMSD) values ​​for these two GluN1 protopolymers with all Cα atoms arranged were [value missing]. Compared to the corresponding domains of the GluN1(A) propolymer, when two GluN1-LBDs are aligned, GluN1(C)-NTD rotates outward by 11.3° and GluN1(C)-TMD rotates inward by 2.8°. Figure 3 B). Furthermore, the integration of the GluN2A and GluN2B subunits endows tri-eNMDARs with asymmetric structural features in each layer, most notably in the crosstalk between the NTD and LBD domains. The NTD-LBD angle of the GluN2A subunit is 10° larger than that of the GluN2B subunit (119.1° vs 109.1°). Figure 3 B). These data indicate that individual GluN1 and GluN2 protozoa are asymmetrically integrated into the tetrameric tri-eNMDAR complex.

[0214] Next, structural analysis was performed on the NTD and LBD heterodimers. Since the divalent ion chelating agent ethylenediaminetetraacetic acid (EDTA) is present in the SEC buffer, this triiso-eNMDAR may be trapped in the ligand-free state of the NTD. Comparison of the conformation of GluN2A-NTD in the tri-eNMDAR with the previous structure shows that it is indeed trapped in the zinc-free structure. Figure 8 C). In the tri-eNMDAR, GluN2A-NTD employs a more open gap conformation than GluN2B-NTD, and the distance between the centroids (COM) of the R1 and R2 lobes is measured ( vs Figure 3 C). When the R1 lobes overlap, the GluN2A-R2 lobes exhibit a 7.3° greater twist conformation than the GluN2B-R2 lobes, indicating that GluN2A-NTD plays a dominant role in the high-gating activity of the three receptors. In contrast, the LBD heterodimers between GluN1-N2A and GluN1-N2B show the smallest conformational difference, with a CαRMSD value of [value missing]. ( Figure 3 C), this may be because both GluN2A- and GluN2B-LBD are trapped in an open-slit conformation of the antagonist (R)-cpp binding. Figure 8 D).

[0215] Furthermore, GluN2-NTDs are located above the LBD intermediate dimer interface and interact with the D1 lobes of GluN1- and GluN2-LBDs, thereby transducing the conformational changes of GluN2-NTDs into the LBD layer. Figure 3D). Disulfide crosslinking at these NTD-LBD interaction interfaces in recombinant NMDARs significantly affects channel activity. Measurements of the COM distance between GluN2-R2 and GluN2-D1 or GluN1-D1 leaflets indicate that the interaction between GluN2A-NTD and the LBD is stronger than the interaction between GluN2B-NTD and the area below the LBD. vs 33.9, vs Figure 4 (D) This is consistent with observations of the Xenopus laevis GluN1-N2A-N2B receptor, indicating a broader interaction between GluN2A-NTD and downstream LBD. Within the LBD layer, the COM distance between the GluN2A-D1 lobule and the adjacent GluN1(A)αE helix directly connected to the ion channel gate is shorter than the corresponding distance between GluN2B-D1 and the GluN1(C)-αE helix. ( vs Figure 4 D). These measurements suggest that the GluN2A subunit may have a greater influence in the NTD-LBD and LBD-TMD transitions of tri-eNMDAR.

[0216] For the tri-eNMDAR TMD, the central ion permeation pathway includes a channel gate composed of four transmembrane helices 3 (M3), a selective filter formed by transmembrane helices 2 (M2), and a hydrophobic vestibular cavity sandwiched in the middle. Figure 3 E and 3F). The periphery of the central gate is surrounded by transmembrane helices 1 and 4 (M1 and M4) of four subunits. Figure 3 E). HOLE analysis showed that ion influx and permeation were primarily limited by the closed-channel grid and the tip of the selective filter (E). Figure 3 F). Next, the arrangement of the four hole-liner M3 spirals was measured, and it was found that they were asymmetrically arranged, with the distance between GluN2A-GluN1(A) being longer than the distance between GluN2B-GluN1(C). vs Figure 3 E). Meanwhile, the distance between the GluN2A-M2 and GluN1(A)-M3 helices is greater than the distance between the GluN2B-M2 and GluN1(C)-M3 helices, as indicated by the C**-C** distance of the corresponding residues. Figure 3 F). These data indicate that the four subunits asymmetrically assemble into tri-eNMDARs, endowing tri-eNMDARs with different kinetic and pharmacological properties than di-eNMDARs.

[0217] Example 5. GluN1-N2B di-eNMDAR and its comparison with tri-eNMDAR

[0218] Although GluN1-N2BeNMDARs develop from GluN2B in the newborn to GluN2A in the adult brain, they still exist and constitute the second group of eNMDARs in the adult cortex and hippocampus. Figure 2 B). Unlike tri-eNMDARs, the overall structure of GluN1-N2B di-eNMDARs exhibits dual symmetry in each layer. Figure 4 A). The most obvious structural difference is the NTD tetramer interface formed by the direct interaction of the two R2 lobes of homologous or heterologous GluN2 subunits. In GluN1-N2B di-eNMDARs, the α5 and α6 helical interactions of the two GluN2B-R1 lobes symmetrically form the tetramer NTD interface. Figure 4 A). Disulfide crosslinking at this interface leads to a significant decrease in channel activation of the recombinant GluN1-N2A and GluN1-N2B dual receptors. In tri-eNMDAR, the α5 and α6 helices of the GluN2B-R2 leaflet are slightly elevated relative to its adjacent GluN2A-R2 leaflet. Furthermore, via the α5-α5 helix ( vs ) and R2-R2 leaves ( vs Figure 4 Assessing the COM distance in A), the NTD tetramer interface is more compact in tri-eNDMARs than in di-eNDMARs. It is speculated that the NTD tetramer interface may be a unique region in which molecules are designed to differentiate the physiological functions of tri-eNDMARs and di-eNDMARs.

[0219] Compared to di-eNMDAR, the GluN2B-NTD in tri-eNMDAR adopts a 6.0° twisted conformation, while the COM distance between R1-R2 lobes remains unchanged. Figure 4 B). By comparing NTD heterodimers, it was found that the contact between GluN1-R1 and GluN2B-R1 leaflets was tighter in tri-eNMDARs than in di-eNMDARs. vs Figure 4B). Magnified view of the NTD heterodimer interface shows that the binding cavity of effendil, a GluN2B-specific negative allosteric regulator, is reduced in the tri-eNMDAR. As GluN2B-α2 approaches GluN1-α3, the GluN2B-R1 lobe of the tri-eNMDAR rotates 5.9° toward the GluN1-R1 lobe, and the COM distance between the two helices is shortened compared to the di-eNMDAR. ( Figure 4 C). This narrowed GluN1-N2B NTD interface may impede the accessibility of ifenprodil, which may explain the difference in pharmacological effects of ifenprodil between di- and tri-eNMDARs, as well as its reduced affinity for the brains of neonates to adult rodents.

[0220] On the LBD and TMD layers, the GluN1-N2B LBD heterodimer in tri-eNMDAR forms a more closed embedding interface than that in di-eNMDAR, and the D1-D1 distance is shorter. vs Figure 4 B). The TMD of GluN1-N2B adopts a classic dual-symmetric gate structure ( Figure 4 D). The global superposition of di- and tri-eNMDAR with the M3 helix Cɑ indicates that the conformational motion mainly occurs in GluN1(C)-M3, rotating clockwise along the central axis in the tri-eNMDAR. Figure 4 D). In summary, the GluN1-N2B dimer unit exhibits conformational variability depending on the environment in which it is integrated into the tetramer. Compared to the conformation of GluN1-N2B, the GluN2B subunit in GluN1-N2A-N2B exhibits a less distorted NTD conformation, a more compact GluN1-N2B NTD and LBD heterodimer conformation, and a tighter M3 interaction with the adjacent GluN1(C) subunit. Figure 4 These results demonstrate that the complexity of eNMDARs with multiple subunits and different conformational domains offers opportunities for designing drugs with subunit selectivity and conformational preference.

[0221] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for assessing the function of endogenous NMDA receptors in brain tissue, characterized in that, The brain tissue is selected from the following group: cortex, hippocampus, or a combination thereof. The method includes the following steps: (a) Obtaining data on the structure and / or quantity of endogenous NMDA receptors (NMDARs) in the brain tissue of a subject, wherein the NMDA receptors include GluN1-N2A-N2B triheteromers; and (b) The structure and / or quantity of the GluN1-N2A-N2B triheteromeric molecule are compared with reference structures and / or quantity values ​​to provide an evaluation of the function of endogenous NMDA receptors in brain tissue.

2. The method as described in claim 1, characterized in that, The evaluation results include: evaluation of cognitive function, assessment of mental state, evaluation of memory function, or a combination thereof.

3. The method as described in claim 1, characterized in that, In step (a), the NMDA receptor further includes: GluN1-N2A diisotetramer, GluN1-N2B diisotetramer, or a combination thereof.

4. The method as described in claim 1, characterized in that, The quantity is selected from the following group: the level of NMDAR in the brain tissue, the relative level R (or relative percentage P) of a single NMDAR in the brain tissue, or a combination thereof.

5. The method as described in claim 4, characterized in that, The relative level R or relative percentage P of a single NMDAR is calculated using the following formula Q1 or Q2: R = Zx / Ztotal(Q1) P = Zx / Ztotal × 100% (Q2) In the formula, R represents the relative level of the single NMDAR; P is the relative percentage of the single NMDAR; Zx represents the level of the single NMDAR in the brain tissue; Ztotal represents the total level of all NMDARs in the brain tissue described.

6. The method as described in claim 1, characterized in that, The quantity reference values ​​include reference contents. In step (b), the relative levels of GluN1-N2A-N2B, GluN1-N2A, and GluN1-N2B are compared with reference contents as follows: GluN1-N2A-N2B 45±3%, GluN1-N2A 35±2%, and GluN1-N2B 20±2%.

7. The method as described in claim 1, characterized in that, Step (b) involves comparing the structure of the GluN1-N2A-N2B triheteromer with a reference structure, including: (i) Compare the centroid distance (COM) (preferably, compare the COM distance between GluN1-R1 and GluN2-R1, between GluN2-R1 and GluN2-R2, and / or the rotation angle between the two leaflets of GluN2-R2); (ii) Compare the structural changes of the GluN1-N2A-N2B triheteromer in the presence of NMDA receptor allosteric modulators; (iii) Compare the structural features of the LBD intradimer interface and the LBD interdimer interface; (iv) Compare the conformational changes of the GluN2B subunit between GluN1-N2B and GluN1-N2A-N2B; (v) Compare the conformational changes of the GluN2A subunit between GluN1-N2A and GluN1-N2A-N2B.

8. The method as described in claim 7, characterized in that, In (iv), compare the following structural features: The detwisting of GluN2B-NTD, the tightness of the formation of GluN1-N2B NTD and LBD heterodimers through the R1-R1 and D1-D1 interfaces, the degree of proximity of GluN1(C)-M3 to GluN2B-M3, or combinations thereof.

9. A device for assessing the function of endogenous NMDA receptors in brain tissue, characterized in that, The brain tissue is selected from the following group: cortex, hippocampus, or a combination thereof. The device includes: (M1) Input module, configured to: input data on the structure and / or quantity of endogenous NMDA receptors (NMDARs) in the brain tissue of a given subject, wherein the NMDA receptors include GluN1-N2A-N2B triheteromers; and (M2) Analysis module, configured to: compare the structure and / or quantity of the GluN1-N2A-N2B triheteromeric molecule with a reference structure and / or quantity, thereby providing an evaluation of the function of endogenous NMDA receptors in brain tissue; and (M3) Output module, which is configured to output the evaluation results.

10. A method for screening drugs, characterized in that, Including the following steps: (S1) In the experimental group, the test compound was administered to the subjects, and data on the structure and / or quantity of endogenous NMDA receptors (NMDAR) in the brain tissue of the subjects were obtained DT1; in the blank control group, the test compound was not administered to the subjects and other experimental conditions were the same, and data on the structure and / or quantity of endogenous NMDA receptors (NMDAR) in the brain tissue of the subjects were obtained DT2. The NMDA receptor includes the GluN1-N2A-N2B triisotetramer, and the brain tissue is selected from the following group: The cortex, hippocampus, or a combination thereof; and (S2) Compare the data DT1 with the data DT2. Specifically, if the quantitative characteristics of GluN1-N2A-N2B triheteromers in the brain tissue of the experimental group are closer to the quantitative reference value compared with the blank control group, it suggests that the tested compound is a candidate drug for improving brain function; and / or In particular, if the structural characteristics of the GluN1-N2A-N2B triheteromer in the brain tissue of the experimental group are closer to the reference structure than those of the blank control group, it suggests that the test compound is a candidate drug for improving brain function.

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