Neuronal Menin Overexpression Rescues Learning and Memory Phenotype in CA1-Specific α7 nAChRs KD Mice

The perturbation of nicotinic cholinergic receptors is thought to underlie many neurodegenerative and neuropsychiatric disorders, such as Alzheimer’s and schizophrenia. We previously identified that the tumor suppressor gene, MEN1, regulates both the expression and synaptic targeting of α7 nAChRs in the mouse hippocampal neurons in vitro. Here we sought to determine whether the α7 nAChRs gene expression reciprocally regulates the expression of menin, the protein encoded by the MEN1 gene, and if this interplay impacts learning and memory. We demonstrate here that α7 nAChRs knockdown (KD) both in in vitro and in vivo, initially upregulated and then subsequently downregulated menin expression. Exogenous expression of menin using an AAV transduction approach rescued α7 nAChRs KD mediated functional and behavioral deficits specifically in hippocampal (CA1) neurons. These effects involved the modulation of the α7 nAChR subunit expression and functional clustering at the synaptic sites. Our data thus demonstrates a novel and important interplay between the MEN1 gene and the α7 nAChRs in regulating hippocampal-dependent learning and memory.


Introduction
Cholinergic transmission in the central nervous system (CNS) serves multiple functions ranging from development [1,2], modulation of excitatory [3] and inhibitory [4] transmission [5,6], central processing of pain [7], food intake, anxiety [8] to learning and memory [9]. Activation of cholinergic synapses in the mammalian CNS mediates synaptic modulation and plasticity [3], underlying learning and memory, specifically in the hippocampus [10,11]. Amongst these nicotinic cholinergic receptors (nAChRs), the α7 subunit has attracted significant attention due to its unique properties, such as high Ca 2+ permeability-induced membrane depolarization [12,13], and its role in cognition, memory, immunity, inflammation and neuroprotection [14,15]. Specifically, α7 nAChRs' role in hippocampus-specific learning and memory and its underlying mechanisms have been the focus of ongoing research [16,17]. Moreover, the perturbation of α7 receptor is linked to several neurological disorders, such as schizophrenia (SZ) [18,19] and Alzheimer's disease (AD) [20]. Despite the importance of various cholinergic functions, our understanding of the development, assembly and maintenance of neuronal nicotinic cholinergic receptors is, however, limited due to its widespread distribution and structural/functional diversity in Hippocampal tissue was isolated from E18 embryos in solution (1 × HBSS containing 10 mM HEPES; 310 mOsm, pH 7.2), and treated with enzyme (Papain (50 U/mL) in 150 mM CaCl 2 , 100 µM L-cysteine and 500 µM EDTA in neurobasal medium (NBM)) for 20 min at 37 • C with 5% CO 2 . NBM supplemented with 4% FBS, 2% B27, 1% penicillin-streptomycin and 1% L-glutamine (GIBCO) was given 3× to wash out the enzyme. Using a trituration technique with fire-polished glass Pasteur pipettes, neurons were then dissociated and plated onto glass coverslips at a density of around 900 cells/mm 2 for achieving lower density cultures maintained in NBM supplemented, as aforementioned. The coverslips used were washed previously with nitric acid and coated with poly-D-lysine (30 µg/mL; Sigma Aldrich, Oakville, ON, Canada) and laminin (2 µg/mL; Sigma Aldrich, Oakville, ON, Canada) in costar 12-well plates (VWR). The neuronal culture media (about 50%, was replaced with NBM supplemented with 2% B27, 1% penicillin-streptomycin and 1% L-glutamine on day 2 of cultures and the neurons were maintained throughout at 37 • C with 5% CO 2 . The neuronal media was changed every consecutive day to maintain the neuronal growth.

Immunocytochemistry and Immunohistochemistry
Immunocytochemistry (ICC) and IHC assays were employed to label proteins of interest as described previously [42]. Neuronal cultures were fixed on DIV 3, 7, 10, 14 and 20, respectively, with 4% paraformaldehyde and 0.2% picric acid (Sigma Aldrich) in 1 × PBS and permeabilized for 1 h with an incubation medium (IM) containing 0.5% Triton and 10% goat serum in 1 × PBS. Negative controls were performed to test the specificity of the antibodies, as described previously [31]. To label proteins of interest, primary antibodies (menin C-terminal epitope (Bethyl Laboratories, A300-105A, Montgomery, TX, USA); menin C-terminal epitope (Santa Cruz, SC-374371, Dallas, TX, USA); α-neurofilament (Novus Biologicals, NB300-222, Littleton, CO, USA); α-synaptotagmin (EMD Millipore, MAB5200); α-PSD-95 (Antibodies Incorporated, 75-028, Davis, CA, USA), were used at 1:500 in IM for 1 h. Secondary antibodies (Alexa Fluor 488, 568, or 680 conjugated goat α-rabbit, α-mouse or α-chicken (Invitrogen, Waltham, MA, USA)) were used at 1:100 in IM for 1 h. α7-nAChR were labelled with Alexa Fluor 555 conjugated α-Bungarotoxin (Invitrogen, B35451) and/or Alexa Fluor 488 and/or Alexa Fluor 647 at 2 µg/mL in IM for 1 h. Neuronal cultures were then subjected to three 15 m washes in 1x PBS after each incubation at room temperature. Cells were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen). For brain slices, sections were exposed to freshly made 0.3% hydrogen peroxide in 0.1% sodiumazide for 15 m to block any endogenous peroxidase activity to avoid background labelling of blood vessels. Slices were then subjected to heat mediated antigen retrieval step in Sodium citrate buffer for 10 min and washed with 1XPBS for 15 min. The rest of the protocol for labelling tissue was the same as mentioned above. The specificity of the antibodies used in this study has previously been confirmed using western blot analysis [31]. All antibodies were, however, further optimized on sagittal and coronal 16 µm thick adult mouse brain slices in the AP Research Lab of Alberta Precision Labs, using high precision, reliable and automatic methods, as described above.

Quantitative PCR (qPCR)
Total RNA was isolated from adult C57BL/6 mice (injected with α7 shRNA AAV and α7 scrambled AAV) hippocampus (CA1 region in some cases) using the RNeasy micro kit (QIAGEN, ON, Canada) according to the manufacturer's instructions. Using the quantitative reverse transcription kit (Superscript Vilo, Thermofischer, Waltham, MA, USA), reverse transcription was performed. For the negative control groups, all components except the reverse transcriptase were included in the reaction mixtures. A LUNA kit was used for qPCR assay, and the primers used were directed to a region of 80-120 bases, and the Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as the housekeeping (reference) gene. Control reactions and those containing cDNA from α7 scrambled cultures and/or hippocampi, α7 KD cultures and/or hippocampus were performed with 1 ng of template per reaction. The running protocol extended to 45 cycles consisting of 95 • C for 5 s, 55 • C for 10 s and 72 • C for 8 s using the QuantStudio setup. The specificity of all PCR reactions was checked by dissociation standard curve analysis graph plot, whereas the assay validation was confirmed by testing serial dilutions of pooled template cDNAs, suggesting a linear dynamic range of 2.8-0.0028 ng of the template. Efficiency values for qPCR primers ranged between 85% and 110% (R 2 = 0.97-1.00). The expression of genes in different treatment groups relative to GAPDH was determined using the Thermofischer QuantStudio™ 3D AnalysisSuite™ software, version 3.1.6.

Confocal Microscopy
Images were obtained using confocal microscopy [44] at 60× magnification on neuronal cultures and brain slices (16 µm), as previously described [42], and fluorophores were excited with 402, 488, 568 and 680 laser wavelengths and emissions collected through 450/50, 525/50 and 700/75 filter cubes for different samples. All imaging parameters, including the field of view size, laser intensity and channel gains, were kept strictly constant amongst relevant samples. Images were collected from 9-12 samples prepared from independent culture sessions. All microscope settings are shown (see Supplementary  Table S1). The fluorescence intensity of all antibodies was measured and quantified using IMAGE J. The fluorescence range was from 0 to 250, where AU < 20 was considered background signal, 20 < AU > 50 was moderate and values >50 were deemed strong throughout the quantification of relevant samples.

AAV Production and Transduction of Neuronal Cultures
α7 shRNA AAV and α7 shRNA Scrambled MEN1 Encoding AAV and GFP Only AAV Small hairpin (sh)RNA-encoding constructs were designed targeting the mouse CHRNA7 gene, which encodes α7 subunits of the nicotinic acetylcholine receptors using the splashRNA design tool [45]. The sequences with the highest "splash" scores were selected for experimental use (see Table 1).

Plasmid Construction and AAV Packaging
The shRNA sequences were incorporated into DNA oligos and were used to PCR amplify the U6 promoter of pAAV-U6-shRNA-CAG-tdTomato using Kapa Hifi DNA polymerase (Roche) according to the manufacturer's instructions. The resulting PCR product was subcloned into the vector that had been digested with MluI and HindIII (see Figure S1a). The correct sequence and insertion of the shRNA sequence were verified by DNA sequencing.
The mouse MEN1 cDNA was obtained by ordering mammalian gene collection (MGC) Clone ID 4189611 from Horizon Discovery (NCBI accession NM_001168490.1). The cDNA sequence was PCR amplified and inserted in place of cre recombinase at the BspEI and HindIII restriction sites in pENN-AAV-hSyn-HI-eGFP-cre (a gift from James M. Wilson (Addgene plasmid #105540; RRID: Addgene_105540)) using the NEBuilder HiFi DNA assembly kit (New England Biolabs, Ipswich, MA, USA) to generate pAAV-hSyn-HI-eGFP-MEN1. The GFP only AAV had the same backbone, but no gene sequences were inserted (see Figure S1b).
Mouse hippocampal cultures were transduced with scrambled shRNA or α7 shRNAencoding AAV on DIV 1 by spinoculation (2 m at 2000 rpm) using a multiplicity of infection of~0.2, and the media was changed after 24 h. tdTomato fluorescence was observed after 48-72 h, and the transduction efficiency of mouse hippocampal neurons was estimated to be~90-94%. α7 nAChR KD was confirmed by qPCR and ICC and IHC.

Stereotaxic Injections in C57BL/6 Mice
Adeno-associated virus (AAV) (200 ng/µL) was administered bilaterally in the CA1 region of C57BL/6 mice through stereotaxic injections, as previously described [46]. The coordinates used for injections were 2 mm behind bregma, 2 mm lateral and 1.6 mm below the dural surface for CA1 [47]. The injections were performed with glass micropipettes with a tip diameter ranging between 10 and 20 µm and injected slowly with a pressure-injection system (see Figure S1c). The animals were given post-surgery care for 5 days, where they were monitored for any discomfort or changes in diet or movement (see Figure S1d). The recovery time post-injection ranged between 3 and 5 weeks, after which the animals were either sacrificed for the validation of the model or were subjected to a contextual fear conditioning behavioural test.

Contextual Fear Conditioning Behaviour Test
Contextual fear conditioning (learning and memory test specific to the hippocampus) was conducted in a chamber with plastic walls and a metal rung floor. The internal dimensions of the chamber were approximately 17 × 17 × 25 cm for mice. The mice behaviour was recorded by a digital video camera directly mounted above the conditioning chamber. The setup was acquired from the ANY-maze Stoelting Co. (Wood Dale, IL, USA) Fear Conditioning System, which automatically detected and quantified the freezing behaviour. On Trial Day 1, the animals were placed into the conditioning chamber and habituated to their surroundings for 2 min. Following the habituation, the mice received 0.5 mA shocks of 1 s duration every 2 m. Once the trial was completed, the animals were returned to their home cage, and the chamber was cleaned with 70% ethanol after each trial.
On trial day 2, the mice were placed in the identical environment (chamber) and were subjected to behavioural analysis as day 1, but the shock was not presented. Each minute, the software recorded the freezing score (freezing episodes and freezing time; freezing percentage) for each animal. After completion of the task, the mice were returned to their home cages. The results were exported in excel and analyzed using PRISM as aforementioned.

Experimental Design and Statistical Analysis
To ensure that all results were reproduceable and replicable, data sets were derived from ≥8 independent experiments, using samples from ≥12 independent cell culture preparations or tissue collected from ≥8 animals. The resource equation method was used to determine the minimum sample sizes for quantitative data. IMAGE J (NIH) software was used for image processing and fluorescence intensity unbiased blinded by acquisition file number. For brain slices, an equal area for the region of interest (ROI) was selected randomly (independent of the size of the tissue), n ≥ 12 every slice per region to minimize biases. The quantification tools were kept constant for all tissues amongst relevant samples.
Statistical analyses were performed using Prism8 version 8.3.1 Graph pad software. The data distribution was analyzed with the D'Agostino and Pearson test of normality [48], Bartlett's test for homoscedasticity and parametric (p > 0.05) or non-parametric (p < 0.05) statistical tests were used as appropriate. Differences in fluorescence intensity for ICC were assessed with the one-way ANOVA followed by a post-hoc Tukey test on IMAGE J, Java 1.8. Significant differences in relative gene expression from qPCR from relevant samples on different DIVs were determined using one-way ANOVA followed by a post-hoc Tukey test and Dunnett's multiple comparison test amongst relevant samples. Significant differences in the degree of colocalization test were determined by a one-sample t-test.

Selective Hippocampal, Neuron Specific KD of α7 nAChRs Differentially Regulates the Expression of the MEN1 Gene during Synaptogenesis and Synaptic Maturation
Previous studies from our lab [30,31,42], and others [39], have shown the MEN1 gene and its encoded protein's roles in synaptogenesis [38], synaptic plasticity [31,49] and learning and memory [40]. Moreover, neuronal MEN1 knockout mice exhibit learning and memory deficits [40]. However, it remains to be determined whether there exists reciprocal feedback between menin and α7 nAChRs in cholinergic synaptogenesis and function. To test this possibility, we sought to determine whether selective KD of α7 nAChRs alters the expression patterns of the MEN1 gene and its encoded protein menin.
To this end, we created a plasmid construct with U6-promoter-driven, α7 nAChRspecific shRNA or its scrambled control together with CMV-tdTomato and packaged it into AAV serotype 9, which has been shown previously to exhibit efficient transduction in mouse brain neurons [50] with the aim of impairing α7 nAChRs expression in the hippocampal neurons. Hippocampal neurons were dissected from C57BL/6, E18 mouse pups, and neuronal cultures were prepared for virus transduction. Primary hippocampal neurons were transduced with α7 shRNA AAV and its relevant scrambled control on day in vitro 1 (DIV 1). The transduced neurons were then imaged every day to monitor the AAV transduction using tdTomato fluorescence as an indicator of transduction efficiency (see Figure 1A,B). On DIV 10 (which represents the period of active synapse formation in cultures) and DIV 20 (the period of synapse maturation in culture) [51], RNA samples were collected from untreated controls, α7 scrambled AAV and α7 shRNA AAV for qPCR analysis and assayed for nAChRs neuronal subunits α2-7 and β2-4, as well as the MEN1 gene. Our results demonstrate that α7 nAChRs scrambled AAV had no significant effect on the expression levels of any of the genes that we examined, including nAChRs neuronal subunits α2-7 and β2-4, as well as the MEN1 gene (see Figure 1C,D; n = 5, 3 independent experiments each, see Supplementary Table S2) relative to untreated control on DIV 10-DIV 20. However, in α7 nAChRs shRA AAV transduced cultures on DIV 10, we observed a 4-fold upregulation of the MEN1 gene, as well as a 2.5-fold upregulation of α5 nAChRs, whereas a significant 12.7-fold downregulation of α7 nAChRs (see Figure 1C, n = 5, three independent experiments each, see Supplementary Table S2a). Interestingly on DIV 20, α7 nAChRs KD neurons exhibited about a 13.9-fold downregulation of the MEN1 gene (see Figure 1D, n = 5, three independent experiments each, see Supplementary Table S2b). These results suggest that there might exist a feedback loop between α7 nAChRs and the MEN1 gene, and as such, underscore the importance of the MEN1 gene regulation via subunit-specific, nicotinic cholinergic receptor function.  Table 1. gene during synaptogenesis and synaptic maturation stage (in vitro). (A) live cell phase contrast (i-iii), (B) tdTomato fluorescence (i-iii); images of untreated control (Ai,Bi), scrambled control (Aii,Bii) and α7 shRNA AAV (Aiii,Biii) AAV transduced hippocampal cultures on DIV 7 (n = 21 images, 7 independent samples each, representative images). Scale bar 20 µm. (C,D) Summary data, fold change gene expression in hippocampal cultures on DIV 10 (C) and DIV 20 (D), relative to untreated control, determined by qPCR (n = 6, three independent experiments each, triplicate replicates). α7 KD upregulated MEN1 expression initially and downregulated later in the synaptic maturation stage. Statistical significance (one-way ANOVA followed by Tukey's multiple comparison test) **** p < 0.0001, ns p > 0.9999. See Supplementary Table S2. Next, we used an ICC fluorescence intensity analysis to verify α7 nAChRs KD in α7 nAChRs shRNA transduced hippocampal neurons and its relevant control (scrambled AAV). Images were acquired from tdTomato-positive neurons using confocal microscopy to compare the α7 nAChRs expression in both groups from DIV 3, 7, 10 and 14, where fluorophore-tagged α-BTX was used to label α7 nAChRs. Our data confirmed a 93% reduction of α7 nAChRs in α7 nAChRs shRNA AAV transduced cultures compared to scrambled controls, which had no significant differences in α7 nAChRs expression compared to the untreated controls (see Figure 2A,B, n ≥ 30 each; DIV 3-14, representative images on DIV 10, see Supplementary Table S3). These data validated a subunit-specific KD of nAChRs α7 protein and indicated an underlying feedback loop that may potentially mediate an initial upregulation followed by downregulation of the MEN1 gene following the α7 nAChRs shRNA-induced KD in neurons.

α7 nAChRs KD in Hippocampal Neurons Perturbs Menin Expression
Having established that α7 nAChRs KD induced the downregulation of the MEN1 gene, we next sought to determine whether the expression patterns of menin protein were altered in α7 nAChRs KD neurons in vitro. To address this question, we labelled α7 nAChR KD neuronal cultures with an antibody that recognized an antigen close to the C-terminus of menin (C-menin antibody) on different DIVs, 3, 7, 10, 14 and 20, and quantified the fluorescent intensity using IMAGE J as previously shown [42]. We observed significant changes in the intensity of puncta exhibiting C-menin antibody fluorescence in α7 nAChRs shRNA-transduced tdTomato-positive neurons compared to its scrambled controls during the synaptogenic period (see Figure 3A; n ≥ 30 each; DIV 3-14, representative images on DIV 10, see Supplementary Table S4). The C-menin fluorescence intensity in these puncta, however, gradually decreased compared with their scrambled controls in matured neurons over time (see Figure 3B; n ≥ 30 each; DIV 3-14, representative images on DIV 20, see Supplementary Table S4). This reduction in the numbers of menin-expressing puncta and the timelines were consistent with the MEN1 gene expression data presented in Figure 1.

α7 nAChRs KD in Hippocampal Neuronal Cultures Altered Synaptic Proteins Assembly at the Pre and Postsynaptic Sites
Menin colocalizes both at the pre and postsynaptic sites with SYT and PSD-95 along with the cholinergic machinery in the mouse hippocampus [42]. Neuron-specific MEN1 knockout exhibits an altered synaptic expression of Synaptotagmin, SYT (presynaptic vesicle protein) and Postsynaptic density protein, PSD-95 [40]. Having established that menin is altered in α7 nAChRs KD neurons, we next sought to determine what would happen to the expression of other synaptic proteins involved in cholinergic synaptogenesis [31]. To address this question, we used α7 nAChR shRNA-transduced neuronal cultures and performed ICC. Specifically, we used a PSD-95 antibody to label the postsynaptic PSD-95, SYT-1 antibody to label presynaptic SYT and fluorophore-tagged α-BTX to label α7 nAChRs on DIV3, 7, 10 and 14, respectively. Consistent with our previously published data [40], the ICC results demonstrated a significant reduction in the puncta exhibiting PSD-95 fluorescence in α7 nAChR KD neurons from DIV 7 onwards (see Figure 4A,B, n ≥ 30 each; DIV 3-14, representative images on DIV 10, see Supplementary Table S5). In contrast to postsynaptic, the puncta-expressing presynaptic protein, SYT significantly increased in tdTomato-positive α7 nAChR KD neurons compared to our scrambled and untreated controls (see Figure 5A,B, n ≥ 30 each; DIV 3-14, representative images on DIV 10, see Supplementary Table S6). Taken together, these results suggest a menin-dependent perturbation of both pre and postsynaptic proteins and underscores the importance of the menin-induced α7 nAChRs role in the assembly of synaptic machinery.   To test whether menin and α7 nAChRs expression are interdependent in the intact mouse hippocampus as observed in the in vitro neuronal cultures, we used two-month-old adult C57BL/6 mice and stereotaxically injected α7 nAChRs or scrambled control shRNA-AAV9 in CA1 coordinates (bilaterally in both hippocampi) (see Figure 6A). The weight and the feeding habits of the animals were closely monitored post-surgery, and there were no significant reported differences between the brain weight between α7 nAChRs KD mice compared to its relevant controls (See Figure S5A,B). Four weeks post-injection, the animals were sacrificed, and the CA1 hippocampal region was dissected for RNA analysis followed by qPCR, where we assayed for nAChRs neuronal subunits α2-7, β2-4, SYT and PSD-95, as well as the MEN1 gene. Our qPCR data confirmed the downregulation of α7 nAChRs, which validated their KD, specifically in the CA1 region. Additionally, our data also showed the downregulation of the MEN1 gene in α7 nAChRs KD neurons, whereas the MEN1 gene expression in the scrambled controls remained unchanged relative to the untreated controls (see Figure 6B, n = 5, three independent experiments each with 3X replicates, see Supplementary Table S7). Interestingly, in the α7 nAChR KD tissues, we also observed a downregulation of PSD-95 and an upregulation of the SYT gene, which further confirmed that the α7 nAChR KD did indeed perturb menin-associated synaptic assembly. Having confirmed that the α7 nAChR and MEN1 genes were indeed downregulated at the gene level, we next prepared brain slices to look for the expression levels of α-Bungarotoxin labelled nAChRs-α7 in the CA1 region using IHC assay. Our IHC results were consistent with the in vitro findings, confirming that indeed α7 nAChR expression was significantly reduced in α7 nAChR KD mice in the CA1 region (see Figures 6C and 7C, n ≥ 25 each; adult mouse brain hippocampi, representative images, see Supplementary Table S8). Taken together, these data propose that an analogous mechanism of a feedback loop might exist between α7 nAChRs and menin protein in the hippocampal neuronal networks. To examine whether menin protein was altered in vivo, as well in α7 nAChRs KD in the CA1 neurons, we employed an IHC assay to analyze IHC fluorescence intensity (IMAGE J) in the synaptic puncta. We labelled hippocampal brain slices with the C-menin antibody, as described earlier. The IHC results from the fluorescent intensity measurement of C-menin were consistent with our in vitro cell culture data (DIV 20), which exhibited decreased C-menin protein expression in α7 nAChR KD mouse brain slices (see Figure 7A,B, n ≥ 25 each; adult mouse brain hippocampi, representative images, see Supplementary  Table S9). Taken together, our in vivo results were consistent with that of the in vitro, and together they underscore the importance of the MEN1 gene regulation via subunit-specific, α7 nAChR function in the hippocampus.
As shown in our in vitro α7 nAChR KD model (see above), both pre and postsynaptic proteins were perturbed in α7 nAChR shRNA-induced neurons; we next sought to investigate whether the synaptic assembly in the intact hippocampus was also altered owing to menin downregulation in α7 nAChR KD neurons. To address this question, we labelled hippocampal (CA1) brain slices with PSD-95 and SYT-1 antibodies, respectively (as aforementioned), and quantified the fluorescence intensity using IMAGE J, as previously established [42]. Intriguingly, we observed a significant reduction in PSD-95 expression in the α7 nAChR KD CA1 region compared to our scrambled controls (see Figure 7C and Figure S2, n ≥ 25 each; adult mouse brain hippocampi, representative images, see Supplementary Table S10) and a significant increase in the SYT-1 fluorescence labelled puncta in α7 nAChR KD CA1 neurons (see Figure 7D and Figure S3, n ≥ 25 each; adult mouse brain hippocampi, representative images, see Supplementary Table S11) consistent with our in vitro findings. Taken together, these data suggest the importance of menin-induced α7 nAChRs clustering in the assembly of synaptic machinery in an intact mouse brain.

Restoring Menin in a7 nAChRs KD Hippocampal Neurons Rescued the Expression of a7 nAChRs and Clustering at Synaptic Sites
Previous studies have shown that the KD of the MEN1 gene using an shRNA approach in the hippocampal neuronal cultures results in a loss of α7 nAChRs clusters at the synaptic sites [31]. Our data presented here demonstrate that α7 nAChRs KD in neurons both in vitro and in vivo downregulate MEN1 and its protein product, menin (as shown above). We next asked whether the clustering of α7 nAChR at the synaptic sites was directly dependent on menin expression at the synapses. To address this question, we designed a construct with a neuron-specific synapsin promoter driven by recombinant MEN1, which was N-terminally tagged with eGFP and packaged into AAV9 to selectively target α7 nAChR KD cultures. We overexpressed exogenous recombinant, GFP-tagged MEN1 encoding AAV in α7 KD cultures by transducing the hippocampal neuronal cultures with MEN1 AAV on DIV1 (see Figure S4). We also employed CA1-specific α7 nAChR KD mice to stereotaxically inject MEN1 encoding AAV with the same coordinates, as aforementioned for our in vivo experimental design (see Figure S4).
We then sought to determine if an overexpression of the MEN1 gene could restore the expression of α7 nAChRs clusters at the synaptic sites. To this end, we used an ICC assay to label the five following groups; (1) α7 nAChR scrambled AAV, (2) α7 nAChR shRNA AAV, (3) α7 nAChR AAV+MEN1AAV, (4) α7 nAChR scrambled AAV+MEN1 and (5) α7 nAChR AAV+GFP AAV, respectively, with α-Bungarotoxin labelled and c-terminal menin antibody to measure the expression of these proteins using fluorescence intensity. Our ICC results demonstrated that an overexpression of MEN1 rescued α-Bungarotoxin-labelled α7 nAChRs in α7 nAChR KD neuronal cultures (see Figure 9B,C, n ≥ 18 each; DIV 3-14, representative images on DIV 20, see Supplementary Table S17). Nicotinic cholinergic receptors' specific subunits of α7 nAChRs clusters were also restored in the CA1 region of α7 nAChR+MEN1 mice, as shown by our IHC data (see Figure 10A and Figure S5, n ≥ 18 each; adult mouse brain hippocampi, representative images, see Supplementary Table S18).
Taken together, our findings suggest menin-dependent clustering of α7 nAChRs and underscore its importance in regulating α7 nAChRs assembly at the synaptic sites.

Overexpression of Exogenous Menin in the α7 nAChRs KD Mice Rescues Hippocampus Dependent Learning and Memory
Next, we sought to determine whether the loss of CA1-specific α7 nAChRs knockdown had any significant impact on learning and memory in these mice. To this end, we first performed a contextual fear conditioning assay (which is specific to hippocampusdependent learning and memory) in α7 nAChRs knockdown mice (see Figure 10B). Our data from α7 nAChRs knockdown mice indicated significant deficits in learning and memory (no difference in freezing episodes between two days) (see Figure 10C, n = 15, see Supplementary Tables S17 and S18). These data specifically demonstrated that the selective knockdown of nAChRs α7 in hippocampal neurons significantly affected learning and memory in freely behaving mice. To test whether exogenous MEN1 encoding AAV could restore this learning and memory deficit in α7 knockdown mice, we then stereotaxically injected recombinant MEN1-encoding AAV in the same coordinates (CA1) as mentioned earlier in the α7 knockdown mice, four weeks post-surgery, and performed the same behavioural assay (contextual fear conditioning) on α7 nAChR shRNA AAV+MEN1AAV mice (see Figure 10C). Remarkably, our data showed significant improvement in learning and memory of α7 nAChR shRNA AAV+MEN1AAV mice compared to its relevant controls (significant increase in freezing episodes on day 2 than day 1) (see Figure 10C, n = 15, see Supplementary Tables S17 and S18). The freezing percentage in all the four groups exhibited the same trend as observed in the freezing episodes (See Figure S6 and Table S18).

Discussion
Menin, the protein product encoded by the MEN1 gene, has been extensively studied for its role as a tumour suppressor. However, in the last two decades, menin's role in CNS specific to synaptogenesis [30], the regulation of synaptic plasticity [39], cognition [40] and depression [41] has come to light. Evidence from our lab demonstrates that menin's role is specific to nicotinic cholinergic transcription, regulation and clustering at the synaptic sites [31,38,42]. These studies provided insights into menin's role specific to the nAChR α7 subunit; however, it has only been shown in vitro, thus limiting their scope. The data presented in this study provides strong evidence that α7 nAChRs KD in hippocampal neurons invokes differential regulation of the MEN1 gene and its encoded protein, menin. Additionally, for the first time, we demonstrated here that restoring menin expression in the α7 nAChRs KD neurons rescues α7 nAChRs clustering and improved hippocampus specific learning and memory [52].
Several approaches, including knockout mouse models, have been used to study α7 nAChRs' function in the CNS [53][54][55]. α7 nAChRs are widely distributed in the whole brain where their expression is found both in neuronal and non-neuronal cells [9,56]; therefore, a global knockout model for α7 nAChRs limits the study for exploring its region-specific function. We sought to KD the α7 nAChRs (as previously shown [8]) specifically in the CA1 region to better understand the CA1-specific role of α7 nAChRs in learning and memory and its underlying molecular mechanisms. For this, we used the shRNA AAV approach [57], which has been shown to effectively KD the expression of the desired gene specific to the targeted region of interest. In our current study, shRNA against the CHRNA7 gene knocked it out in approximately 93% of hippocampal neurons, consistent with the successful AAV approaches used in other studies [57].
The hippocampus is a complex brain structure, which is the fundamental foci for learning and memory in the CNS [58,59], and its atrophy has been linked to memory impairment in AD patients [52].
Contextual fear conditioning, not cued fear conditioning (which is amygdala dependent) [60], is a hippocampus-dependent Pavlovian conditioning test [61], which has been used in past studies [62,63] for hippocampus-specific learning and memory [64]; therefore, we specifically tested for contextual fear conditioning test in our α7 KD model. Even though there are other behavioural tests to explore the role of the hippocampus in learning and memory [65,66], memories tested through these behavioural assays have been shown to have influences from other brain regions [67] as well. Recent studies have demonstrated differential roles of the hippocampal regions, Cornu ammonus, CA1 and CA3 in contextual learning and memory [68], whereas CA1 has been found to be necessary for encoding contextual learning and memory and its retrieval in mammals [69][70][71]. Cholinergic transmission modulates synaptic plasticity, which is required for learning and memory [9,10] specific to the hippocampus. Specifically, α7 nAChRs have been shown to play a role in cognition and learning and memory specific to the hippocampus [3,72]. In the present study, our data from the CA1-specific α7 nAChR KD mice demonstrate deficits in learning and memory using contextual fear conditioning test consistent with past findings, which emphasizes α7 nAChRs role in learning and memory [3,10]. Past studies have shown that menin deletion in neurons emphasizes its role in contextual learning and memory but not in cued learning and memory [40]. Intriguingly, our data showed that overexpressing MEN1 in CA1-specific α7 nAChR KD neurons improves the contextual learning and memory phenotype compared to its relevant control. These findings suggest menin-dependent α7 nAChRs' role in learning and memory, although an interesting find, the mechanisms underlying their association requires further detailed exploration. One limitation of our results could be that we only overexpressed exogenous menin specifically in neurons, whereas α7 nAChR-stimulated hippocampal-specific learning and memory are also affected by inputs from non-neuronal cells [61,73]. Our findings thus specifically accentuate the role of menin-dependent neuronal α7 nAChRs in learning and memory. Both menin and α7 nAChRs have been shown to be localized in glial cells [42,73]; therefore, the mechanisms underlying their roles in learning and memory through glial interaction need to be further explored.
In the CNS, PSD-95 was previously thought to play a role in the assembly of glutamatergic receptors [74] and maturation of excitatory synapses specifically [75]; however, recent studies have highlighted its association with α7 nAChRs [76,77]. Intriguingly, our results from α7 nAChRs KD in primary hippocampal cultures, as well as the CA1 hippocampal brain slices, showed a reduction in the expression of PSD-95 puncta at the synapses, suggesting a bidirectional signalling that might be in play between these two proteins. Whereas PSD-95 at the postsynaptic sites was significantly reduced, we observed an upregulation of SYT (a presynaptic protein) in the α7 nAChRs KD cultures and hippocampal brain slice. Past studies on NDD pathophysiology have shown that PSD-95 is downregulated [78,79], whereas SYT is upregulated [80,81] in the hippocampus of dementia and AD models, consistent with our findings from α7 nAChRs KD. The MEN1 knockout study by another group has shown that MEN1 deletion in neurons upregulated SYT, whereas downregulated PSD-95 expression [40]. One possible reason for this perturbation of synaptic proteins in α7 nAChRs KD could be as a result of the ablation of menin expression in α7 nAChRs KD neurons, which could be a potential mechanism for AD pathophysiology; although this possibility is of significant interest, mechanisms underlying this phenomenon warrant further investigation.
Previous studies from our lab have shown that menin regulates α7 nAChRs transcription and clustering in invertebrate [30] and vertebrate neurons [31]. Knocking down the MEN1 gene in hippocampal neurons perturbs the clustering and expression of nAChRs subunits, specifically α7 nAChRs [31], in vitro. In the present study, we have demonstrated, for the first time, that in the α7 nAChRs KD primary hippocampal neurons, MEN1 is initially upregulated on DIV 10, whereas on DIV 20, it is significantly downregulated. These observations suggest that an initial upregulation of MEN1 may be due to a compensatory feedback mechanism of the cell as menin protein is a transcriptional regulator and is involved in the transcription and clustering of α7 nAChRs. In in vitro studies, DIV 7-10 epitomizes the stage of active synaptogenesis and neuronal growth [51]. The ablation of α7 nAChRs provokes intensified menin expression, which suggests menin's role in cholinergic synaptogenesis and compensatory action to promote neuronal growth [40]. Subsequently, on DIV 20, when the synapses are matured, and neuronal density is constant [51], our data shows that menin expression was significantly attenuated in α7 nAChRs knockdown hippocampal neurons. These findings could be indicative of the exhaustion of the intracellular machinery of the cell due to the overwhelming escalation in menin expression, which in turn might have led to negative feedback and downregulation of the menin protein [82]. As menin is involved in many intracellular signalling cascades and cell-cell interactions, its constant upregulation would disturb the cell's homeostasis and compromise other signalling pathways [36]. Nicotinic cholinergic receptor-specific subunit α7 is highly permeable to Ca 2+ ions [1,83], and their mutation has been shown to impair Ca 2+ permeability of the neuron [84]. As shown by previous studies [30,85], MEN1 s regulation is through neurotrophic factor-mediated activity-dependent mechanisms. Taken together, we can speculate that long-term reduction of MEN1 in α7 nAChRs KD neurons could be through Ca 2+ -dependent activity regulation. Although these speculations are of great interest, the mechanisms underlying these possibilities require further investigation.
Our data demonstrated that the restoration of α7 nAChRs by menin overexpression in the α7 nAChRs KD neurons, which suggests the possibility of menin's involvement in the intracellular signalling of α7 nAChRs subunit-specific transcription, as menin has been reported to be a transcriptional regulator of α5 nAChRs [31], as well as other proteins in the cell [36]. Overall, these outcomes emphasize the multifaceted transcriptional networks underlying MEN1 induction and gene targets that are transcriptionally activated or repressed by menin [86]. Our data also suggests menin's role as a potential candidate as a scaffolding protein for α7 nAChRs amongst the other prospective chaperones [29,34,87] reported for α7 nAChRs, as the synaptic puncta clustering of α7 nAChRs augmented significantly in exogenous menin overexpressed α7 nAChR knockdown neurons. One of the explanations for MEN1-induced restoration of α7 nAChRs could be through the phenomenon of homeostatic synaptic scaling [88], which is a major feedback mechanism seen in other synaptic receptors as well, such as AMPARs [89,90] and mGLuR1 [91] for glutamatergic synapses. Cholinergic homeostatic synaptic plasticity [92] has been shown to induce regulatory responses via transcriptional activation of the K v 4/Shal gene, indicating a receptor-ion channel system coupled for homeostatic modulations in neurons [93]. Taken together, these studies suggest that a similar mechanism of action could underlie MEN1 induced restoration of loss of α7 nAChRs, hence re-establishing the cholinergic homeostasis. One important thing to consider is that adult neurogenesis [94,95] occurs in hippocampal neurons, and the increase in α7 nAChRs receptors and clustering could be due to the formation of new neurons; however, all these results were measured specifically in tdTomato-positive neurons (α7 nAChR knockdown), which filters any increase in nascent neurons in the hippocampus.
Overall, we have shown that α7 nAChR knockdown, both in vitro and in vivo, downregulates menin expression in neurons. Rescue by exogenous menin expression not only restores the α7 nAChRs transcription but also improves the α7 nAChR receptor clustering at the synaptic sites in the α7 nAChR knockdown neurons. The learning and memory impaired in α7 nAChR knockdown mice are rescued by menin overexpression specific to the CA1 region of the mouse hippocampus. Nicotinic cholinergic specific subunit α7 nAChRs are known to be perturbed in AD hippocampal pathology [20,96], schizophrenia [97] along with other synaptic proteins perturbations [96]. Ultimately, our results indicate a menin-dependent regulation of α7 nAChR expression and clustering, which might play a role in α7 nAChRs-related NDD pathophysiology.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.