Menin Reduces Parvalbumin Expression and is Required for the Anti‐Depressant Function of Ketamine

Abstract Dysfunction of parvalbumin (PV) neurons is closely involved in depression, however, the detailed mechanism remains unclear. Based on the previous finding that multiple endocrine neoplasia type 1 (Protein: Menin; Gene: Men1) mutation (G503D) is associated with a higher risk of depression, a Menin‐G503D mouse model is generated that exhibits heritable depressive‐like phenotypes and increases PV expression in brain. This study generates and screens a serial of neuronal specific Men1 deletion mice, and found that PV interneuron Men1 deletion mice (PcKO) exhibit increased cortical PV levels and depressive‐like behaviors. Restoration of Menin, knockdown PV expression or inhibition of PV neuronal activity in PV neurons all can ameliorate the depressive‐like behaviors of PcKO mice. This study next found that ketamine stabilizes Menin by inhibiting protein kinase A (PKA) activity, which mediates the anti‐depressant function of ketamine. These results demonstrate a critical role for Menin in depression, and prove that Menin is key to the antidepressant function of ketamine.


Neuron and HEK293T culture procedures
Primary neurons were dissected from timed-pregnant females at E16.5.Briefly, brain cortices were dissected from pups of varying genotypes.The meninges were removed, and cortical tissue was dissociated by enzymatic digestion.Isolated primary neurons were plated on poly-D-lysine coated dishes, and cultured in Neurobasal medium supplemented with B27 (Gibco)/1% penicillin/streptomycin (Invitrogen) and maintained in a 5% CO2 incubator at 37°C.HEK293T was purchased from ATCC for a vial (2 × 10 6 )(Cat#CRL-3216, RRID:CVCL_0063).HEK293T was cultured in DMEM supplemented with 10% FBS and maintained at 37°C in humidified air containing 5% CO2.

ChIP
ChIP procedures were performed followed the manufacturer's instructions Millipore).Briefly, primary neurons from control and NcKO mice (dissected at E16.5) were cultured for 10-12 days and fixed for 10 min at room temperature with media containing 1% formaldehyde and quenched with 125 mM glycine for 5 min.
Fixed homogenates were washed twice using ice-cold PBS containing protease inhibitors.Fixed nuclei were pelleted at 4 min at 2,000 rpm and re-suspended in SDS Lysis Buffer (catalog #20-163), where chromatin was sheared using a SCIENTZ ultrasonic apparatus set to 28% power for 14 cycles of a 4.5 s sonication and a 9.0 s resting stage on ice.The sonicated cell supernatant was diluted 10-fold in ChIP dilution buffer (catalog #20-153) and pre-cleared using protein an Agarose/Salomon Sperm DNA (catalog #16-157).After brief centrifugation, ChIP was performed using 3 mg Menin antibody (A300-105A; Bethyl), H3K27me3(Ab8580; Abcam) or normal rabbit IgG (H2615; Santa Cruz) antibody incubated overnight, followed by enrichment using protein A Sepharose beads for 4 hr.Beads were washed three times with four different buffers (low-salt immune complex wash buffer, high-salt immune complex wash buffer, and LiCl immune complex wash buffer) and one wash with TE (50 mM Tris HCl, 10 mM EDTA).Chromatin was eluted by agitation at 65℃ for 20 min in TES (TE plus 1% SDS) and reverse crosslinked overnight at 65℃.Chromatin was subjected to RNase and Proteinase K treatment, followed by DNA purification by phenol chloroform extraction and ethanol precipitation.DNA pellets were re-suspended in 10 mM Tris and subjected to qPCR (480; Roche).

Electrophysiological recording
Whole-cell patch clamp recordings were obtained from pyramidal neurons in the cortex of G503D mice and littermate control mice; NcKO and littermate control mice using 4-8 MΩ borosilicate glass pipettes (Harvard Apparatus) containing (in mmol 1 - 1 ): 8.0 NaCl, nominally 0.0001 CaCl2 (without Ca 2+ buffering, the concentration cannot be precise), 10.0 Na-Hepes, 130 potassium gluconate, 1.0 MgCl2, 0.3 NaGTP, and 2.0 NaATP (pH adjusted to 7.4 with methanesulfonic acid; 295-300 mosmol 1 − 1 ), unless otherwise specified.Glass pipettes with an Ag-AgCl electrode were connected to a CV-4 headstage and Axopatch-1D amplifier with a Digidata 1200 interface (Axon Instruments) and positioned within the tissue using a motorized patch-clamp micromanipulator.Cell-attached 4-10 GΩ seals were obtained using the blind-patch technique as described elsewhere.Upon seal formation, the whole-cell patch configuration was achieved by gently applying brief suction.Typical whole-cell access resistance (Ra) was 5-30 MΩ and whole-cell leak was below 20 pA.Ra and leak were checked prior to each experiment and if Ra or leak changed by more than 20% or 30 pA, respectively, during a recording then the recording was discarded.Miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) were recorded in the presence of tetrodotoxin (500 nmol L -1 ).We recorded mEPSCs and mIPSCs at holding potentials of -70 and 0 mV, respectively, in the same cell (3 minutes each; n＞20cells/group).

RNA-Sequencing analysis
Cortex of G503D mice and littermate control mice were isolated to obtain RNA which was then used for RNA sequencing analysis.cDNA library construction and sequencing were performed by the Beijing Genomics Institute using Illuminate platform.High-quality reads were aligned to the mouse reference genome using Bowtie 2. Expression levels for each of the genes were normalized to fragments per kilobase of exon model per million mapped reads (FPKM) using RNA-seq by Expectation Maximization (RSEM).Genes with ≥ 2-fold change and P<0.05 were considered statistically significant.
PcKO and control mice were crossed with Ai14 TD tomato reporter mice to obtain 1-month-old off spring mice whose PV neurons with fluorescence.After washing, the freshly peeled brain tissue of the mice was cut into 8 sagittal sections and transferred into the gentle Macs C tube (Meltenyi Biotech).Single cell suspensions of the above mice brain were separated according to the manufacturer's instructions.Then flow cytometry was used to isolate PV neurons of PcKO mice and control mice.Isolated RNA was subsequently used for RNA-seq analysis.cDNA library construction and sequencing were performed by the Beijing Genomics Institute using BGISEQ-500 platform.High-quality reads were aligned to the mouse reference genome using Bowtie 2. Expression levels for each of the genes were normalized to fragments per kilobase of exon model per million mapped reads (FPKM) using RNA-seq by Expectation Maximization (RSEM).We identified DEGs (differential expressed genes) between samples and performed clustering analysis and functional annotation.Genes with ≥ 2fold change and P<0.05 were considered statistically significant.Pathways overrepresented by DEGs were annotated in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database.

Forced Swimming Test
The forced swim test (FST) was used to assess depressive-like behavior.Mice were placed in a container filled with water that eventually resulted in immobility, reflecting behavioral despair.Water (23 ± 1°C) was placed in a transparent acrylic cylinder bath (10cm in diameter, 20cm in height) filled to a depth of 13cm.Mice were placed in the water for six minutes using a video tracking system (Smart 3.0).
Immobility duration (%) within the final 5 minutes of testing was recorded.

Tail Suspension Test
The tail-suspension test was used to assess the efficacy of antidepressants in mice.
Mice were suspended by their tails from an acrylic bar (15cm in diameter, 30cm in height) for six minutes and monitored using a video tracking system (Smart 3.0).Escape-related behavior was assessed, where immobility duration (%) during the 6 minutes suspension period was recorded.

Sucrose Preference Test (SPT) and Sucrose Consumption Test (SCT)
Animals were first trained to consume a 1% sucrose solution from two differing bottles (48 hours before the formal experiment).Twenty-four hours later, the animals were allowed free access to 1% sucrose and water from two differing bottles.To avoid bottle side preference, the two bottles were switched once.The amounts in the two bottles were measured after 24 hours and sucrose preference was calculated according to the following formula: sucrose preferences (%) = sucrose consumption / (sucrose + water consumption) ×100%.

Morris Water Maze
The water maze used in this study comprised a circular tank 120 cm in diameter with a platform filled with tap water at a temperature of 22 ± 2°C.Different shapes were posted along the walls of the tank, which served as spatial reference cues.A camera was mounted above the maze to record the swimming traces in the water maze.
During the acquisition trials, the platform was submerged 1-2 cm below the water surface, mice were place into the maze at one of four points (N, S, E, W) facing the wall of the tank.Mice were allowed to search for a platform for 60 s.If a mouse failed to find the platform, it was guided to the platform and maintained on the platform for 10 s.Four trials a day were conducted with at an intermission of 1 h minimum between trials.Escape latency which indicated spatial learning memory acquisition, was recorded for each trial.On day 7, the platform was removed and a probe test was conducted.The percentage time spent in each of the four quadrants and the number of target (platform) area crossings, mean speed, total distance was recorded.

Rotarod Test
Mice were placed on a stationary rotarod (AccuRotor Rota Rod Tall Unit, 63-cm fall height, 30-mm diameter rotating dowel; Accuscan, Columbus, OH).The dowel was then accelerated to 60 rpm min -1 , and the latency to fall (in seconds) was recorded.The procedure was repeated over 4 consecutive trials, which was averaged to yield the daily latency to fall for each mouse.If an animal fell off the rotarod rapidly (e.g., due to inattention or slips), they were placed back on the rotarod for an additional trial, and the latency was not included in the daily average.The entire procedure was repeated for 2 additional days for a total of 3 days.In addition to the average latency across the 4 trials per day, the maximum latency to fall per day was also analyzed.

Open Field Test
To explore locomotion and spontaneous activity, we characterized mouse behavior as they freely explored an open-field plastic chamber (40-cm width ×40-cm length × 30-cm height) using a video tracking system (Smart 3.0).2-months old mice were placed in this arena for 10 min., and total distance and time spent in the center region (20cm×20cm) was recorded.

Elevated Plus Maze
The

Western blotting
Cultured cells and mouse brain tissues (include cortex and hippocampus) were homogenized in lysis buffer (RIPA) on ice for 40 min, and subsequently centrifuged at 12,000 rpm for 10 min at 4°C.Sample containing 30 g of protein was separated using 10% SDS-PAGE gels.Proteins were transferred onto PVDF (polyvinylidene difluoride) membranes in an ice-cold buffer (25 mM TrisHCl,192 mM glycine,and 20% methanol) by electro transfer for 1.5 h.Immunoblots were probed with indicated antibodies.Goatanti secondary antibodies were purchased from Millipore (#AP132P, #AP124P).
Quantification of band intensities were normalized to β-actin or GAPDH.

Quantitative RT-PCR
Total RNA from animal tissues and cells were isolated using Trizol reagent according to the manufacturer's instructions (Invitrogen).Reverse transcription was performed using ReverTra Ace qPCR RT Master Mix (Toyobo,.RNA concentrations were adjusted to 1.2 g L -1 in nuclease-free water, and total RNA was reverse-transcribed in a 20 L reaction volume.cDNA was amplified by real-time quantitative RT-PCR using SYBR Green (Roche) reagent.Samples were assayed in triplicate and GAPDH was used as an internal control. Pimer sequences used in this study are as follows:

Immunofluorescence
Mouse brain sections were washed three times with PBS and antigen retrieval was performed using citrate buffer (pH 7.0); samples were then permeabilized and blocked in PBS containing 0.5% Triton X-100 and 10% normal goat serum at room temperature for 1 h.Sections were incubated with primary antibodies in blocking buffer overnight at 4°C.After washing, secondary antibodies were added to the blocking buffer and incubated for one hour.Samples were then washed, and counterstained with DAPI.
Images were acquired using a Nikon confocal microscope.

Cycloheximide chase assay
GFP-tagged MEN1 or MEN1 mutation constructs were transfected into HEK293T cells treated with cycloheximide (CHX) at 200 mg mL -1 at different time points.
Proteins were extracted and subjected to western blotting.Turnover of WT-Menin/Menin variants were quantified using Image J.
Figures S1 to S11

Figure
apparatus comprised two opposing open arms (50×10 cm) and two opposing closed arms with roofless gray walls (40 cm) connected by a central square platform and positioned 50 cm above the ground.Mice were placed in the open arms facing an open arm, and their behavior was tracked for 5 min with an overhead camera and Smart 3.0 software.Time spent in the open arms (%) was reported.