Activity-dependent synthesis of Emerin gates neuronal plasticity by regulating proteostasis

Laboratory animals

All animal procedures were conducted with male and female mice according to protocols approved by the Institutional Animal Care and Use Committee (IACUC# 08–0082). Mouse lines EMX1^IRES–Cre (EMX^Cre, JAX 005628), vGat^IRES–Cre (vGat^Cre, JAX 16962) and R26^{lsl–GFP-mMetRS} (JAX 028071) were obtained from Jackson Laboratory. Genotyping was conducted according to protocols published on Jackson Lab websites. To generate homozygous EMX^Cre/WT-mMetRS^KI/KI and vGat^Cre/WT-mMetRS^KI/KI mice, we backcrossed male mMetRS^KI/KI to female EMX^Cre/WT-mMetRS^KI/WT, and male vGat^Cre/WT-mMetRS^KI/WT to female mMetRS^KI/KI, respectively, to reduce germline recombination as reported previously.⁸¹ Germline recombination was further tested with PCR for the backcrossed offspring with primers listed below. This primer pair detects the non-recombined R26 insertion allele LSL-GFP-mMetRS in mouse ear tissue, where the forward primer recognizes the LoxP-STOP-LoxP cassette.

Forward: 5′-ctcatgcgttgggtccac-3’

Reverse: 5′-ccggtggtgcagatgaactt-3’

Primary neuronal cultures from neonatal mice

Primary neurons from male and female C57BL/6 P0 mouse pups were prepared as described below and used at 12 DIV (days in vitro).

Primary neuronal cultures from neonatal mice

Primary neurons from C57BL/6 P0 mouse pups were cultured with Neurobasal-A media (ThermoFisher, Cat#10888022), supplemented with 2% B27 (ThermoFisher, Cat#17504044), 1% Pen Strep (P/S, ThermoFisher, Cat#15140–122), and 1% GlutaMax (ThermoFisher, Cat#35050061). Complete culture media was pre-warmed to 37°C before use.

P0 mice were euthanized and dissected in 1% P/S-HBSS (HBSS, ThermoFisher, Cat#14175095) to obtain cortical tissue with meninges removed. Cortex tissue was then incubated in digestion buffer (complete culture media with 6% (v/v) papain (Worthington Biochemical, Cat#LS003126) and 750U/μL DNase I (Sigma-Aldrich, Cat# 4716728001)) at 37°C for 20 min, with periodic agitation. Then, digested tissue was sequentially gravity-precipitated in 20% FBS-HBSS (FBS, Peak Serum, Cat#PS-FB3) and then HBSS. Subsequently, precipitated pellets were gently triturated in dissociation buffer (1x HBSS with 14.44 mg/ml MgSO₄ (Sigma-Aldrich, Cat#M2643)). Supernatant was then carefully taken and added to 20% FBS in HBSS and centrifuged 3 min at 200 rcf. Supernatant was discarded, and cell pellet resuspended in the complete culture media. Neuron density was measured with Countess cell counter (The Countess 3 FL), and neurons were seeded according to the plate format listed below. Multiwell plates were pre-coated with 0.05 mg/ml poly-D-lysine (MilliporeSigma, Cat#A-003-E) in Borate buffer (1.25g Boric acid, 1.9g Sodium tetraborate in 400mL ddH₂O, pH adjusted to 8.5) overnight (O.N.) at 37°C. 1% P/S-HBSS was added to space in between wells to prevent excessive media evaporation. For 96-well plates, wells on the outer rim were not seeded and filled with 1% P/S-HBSS to avoid edge/corner effects.

The cultures were maintained in the complete culture media in a 37°C incubator with 5% CO₂. At DIV3, FUdR (5-Fluoro-2′-deoxyuridine, 400x stock solution was made with 2 mg/ml FUdR (8mM) and 50 mg/ml uridine (4mM)) was added to culture media. At DIV4, neurons were transduced with AAVs based on experimental designs. At DIV12, cultured neurons were further treated with labeling, stimulation, or directly harvested for subsequent procedures according to experimental purposes. We estimated the ratio of excitatory to inhibitory neurons in the cultures to be 1–1.5:1 based on analysis using two approaches: 1. We counted the numbers of mRuby+ neurons based on vGAT and CaMKIIa induced Ruby expression. This indicated an excitatory: inhibitory ratio of roughly 1:1. 2. We counted CaMKII+ and vCAT+ neurons based on immunolabeling and this indicated an excitatory: inhibitory ratio of 1.5:1.

Immunohistochemistry

Animals were anesthetized with isoflurane (Covetrus, Cat#11695067772) and perfused with 1x PBS (ThermoFisher, Cat# 70011044) followed by 4% PFA (Electron Microscopy Sciences, Cat#15710-S) in 1x PBS. Brains were dissected and post-fixed in 4% PFA at 4°C O.N. and then switched to 1x PBS. 90μm coronal sections were prepared on a vibratome (Leica VT-1000s). Coronal sections between bregma −2.7mm and −3.64mm were selected for analysis of primary visual cortex. Multiwell dishes containing the sections were wrapped with foil to protect samples from light hereafter, as well as during the whole staining procedure. Sections were stored for up to 4 months at 4°C in 0.5% sodium azide (Sigma-Aldrich, Cat#S2002) in 1x PBS to prevent bacterial growth. All incubation steps hereafter were conducted on a horizontal shaker to provide mild agitation. Sections were incubated in antigen retrieval buffer (100 mM sodium citrate (Sigma-Aldrich, Cat#S-4641), 0.05% Tween 20 (Fisher Bioreagent, Cat#BP337100) in 1x PBS, pH = 6.0) for 5 min at RT, followed by brief boiling, then incubated in antigen retrieval buffer for additional 5 min at RT. Sections were then washed 3 times in 0.01% PBS-Tx (1xPBS with 0.01% Triton X-100 (Sigma-Aldrich, Cat#X100)) for 10 min at RT, followed by permeabilization in 0.05% PBS-Tx100 for 1h at RT. Sections were then blocked in IHC blocking buffer (4% bovine serum albumin (BSA, Fisher Bio-reagent, Cat#BP1600100), 3% normal donkey serum (Jackson Immuno Research Labs, Cat#017–000-121), 0.1% Triton X-100, and 0.5% sodium azide in 1x PBS) for 1h at RT. Subsequently, sections were incubated with primary antibodies diluted in IHC blocking buffer O.N. at 4°C (see concentrations in Table). The next day, sections were washed 3 times with 0.01% PBS-Tx100 for 10 min at RT, followed by incubation with secondary antibodies diluted in IHC blocking buffer for 2h at RT, then washed 3 times with 0.01% PBS-Tx100 for 10 min at RT. Finally, sections were mounted on glass slides (Fisher Scientific, Cat#22–034979) with PVA-DABCO (Sigma-Aldrich, catalog #10981) and sealed with a coverslip (VWR, Cat#16004–312). Processed slides were stored at 4°C within a week until imaging.

Immunocytochemistry

Neuronal cultures in 96-well plates were transferred from the incubator to room temperature (RT). To protect from light, the plates were covered with foil hereafter. Multichannel pipette was used to increase reproducibility of the staining procedure. Pipetting should be as gentle as possible to avoid disturbing the cell layer. Cultures were fixed in 4% PFA in 1x PBS for 20 min at RT, followed by rinsing in 1x PBS. Cultures were then treated with blocking and permeabilization buffer containing 3% BSA and 0.1% Triton X-100 in 1x PBS for 30 min at RT. After this, cultures were then incubated in primary antibodies diluted to the optimal concentration in ICC blocking buffer (3% bovine serum albumin in 1x PBS) at RT for 2h (see concentrations in Table). After a rinse with 1x PBS, cultures were then incubated with the secondary antibodies diluted 1:500 in ICC blocking buffer at RT for 1h. Following a final wash in 1x PBS, the plate was ready for imaging. Immunolabeled cultures can be stored at 4°C within a week before imaging.

Western blots

Cultured primary neurons were extensively washed with 1x DPBS and lysed in the dish with RIPA buffer (G-Biosciences, Cat#786–490) and 1x protease inhibitor (ThermoFisher, Cat#A32963). Dissected brain tissue was lysed and homogenized in 1x DPBS with a hand-held tip sonicator (Fisher Scientific, sonic dismembrator model 100). Protein concentration of the homogenate was determined using either BCA protein assay kit (ThermoFisher, Cat#23225) or DC protein assay kit (Bio-Rad, Cat#5000111). Protein extract with ANL or AHA labeling was clicked with biotin-alkyne described above. 6x Laemmli loading buffer (Boston BioProducts, Cat#BP-111R) was added to protein sample to reach 1x final concentration, vortexed and then boiled for 10 min at 100°C.

Western blots were done using MiniPROTEAN electrophoresis cell and compatible gradient precast gels (Bio-Rad, Cat#456–1096), at 120V constant voltage for 40–50 min. Proteins were transferred to nitrocellulose membranes using Trans-Blot Turbo Transfer System (Bio-Rad, Cat#1704150) and the compatible transfer kit (Bio-Rad, Cat#1704158).

Western blot membranes were subsequently stained with ponceau (Sigma-Aldrich, Cat#09189) for 5 min at RT. After scanning the ponceau signal on a scanner, the membrane was blocked with 5% blocking reagent (Bio-Rad, Cat#1706404) in 0.05% TBS-Tw20 for 1 h at RT, then incubated with primary antibodies diluted in WB blocking buffer (2.5% bovine serum albumin in 0.1% TBS-Tx100) O.N. at 4°C (see concentrations in Table). The next day, the membranes were washed 3 times for 10 min with 0.05% TBS-Tw20, followed by incubation with secondary antibodies diluted in WB blocking buffer for 2h at RT. Membranes were washed again in 0.05% TBS-Tw20 3 times for 10 min at RT. Membranes were then incubated with Pierce ECL (ThermoFisher, Cat#32106) for 4 min and developed onto light-sensitive film (Ece Scientific Co, Cat#E3018) for various amounts of time in a darkroom. Western blot data were analyzed in ImageJ using the lane analysis function.

Noncanonical amino acid labeling of newly-synthesized proteins

Bio-orthogonal noncanonical amino acid tagging (BONCAT) metabolically labels the nascent proteome by incorporation of noncanonical amino acids. To identify VE-dependent nascent proteome dynamics in excitatory and inhibitory visual cortical neurons, we dark-housed male and female critical period (P28) and adult (P56) mice for 3 days and delivered ANL by i.p. injection immediately before either 4h of VE or 4h in total darkness (DK). We collected the visual cortex 6h later and prepared samples for BONCAT and DiDBiT (Figure 1A).

Dark housing and visual experience

Mice were reared normally under a 12/12 light-dark cycle before being subjected to this protocol. First, mice were dark housed in a light and sound-proof chamber with ventilation and temperature/humidity control for 72h. Mice then received an i.p. injection of ANL, as described above, under dim red light. The DK group was returned to their cage inside the dark housing chamber until tissue collection. The VE group was placed inside a visual experience arena with transparent walls and running wheels, also inside of the dark-housing chamber. 1h after i.p. ANL injection, 6 video tablets were placed to surround the arena, and played a black-white visual noise video to provide visual stimulation including motion and contrast.⁸² Freely moving mice were exposed to the visual experience for 4h and then housed in darkness for another 5h. This 5h period in darkness allows the immediate-early wave of visual experience responses to pass in order to capture synthesis and accumulation of newly synthesized effector proteins and maximize signals for proteomic analysis. Tissue collection was then performed under dim red light, where mice were deeply anesthetized with isoflurane and euthanized by cervical dislocation. Brains were then dissected, snap-frozen in isopentane (Millipore Sigma, Cat#270342–2L) incubated on dry ice and stored at −80°C before subsequent processing.

BONCAT

BONCAT was performed as described previously,⁸³ following the click recipe below and scaled proportionally to protein amount. Proteins were homogenized in DPBS and SDS solution was added to reach 0.05% final concentration to help release lipid-associated proteins. The assembled reaction was then thoroughly vortexed and incubated at RT for 1h. An extra vortex step was performed at 30 min. The click reaction sample was stored at −20°C for up to a week before further analysis.

Cell type-specific nascent proteomic analysis with DiDBiT in mouse visual cortical tissue

Mouse primary visual cortex was micro-dissected on ice. Specifically, a 3mm-thick section was made from lamda toward anterior using a mouse coronal brain matrices (Ted Pella, Cat#15050). Visual cortex tissue was then homogenized in 200μL 1x DPBS using a hand-held tip sonicator. Protein concentration was determined using the BCA protein assay kit.

Visual cortices were dissected and pooled from 5 mice treated under the same condition to accumulate 10mg protein as starting material for subsequent preparation of each MS sample. The following protocol was adapted from DiDBiT and other protocols.^41,83 Specifically, VE and DK samples were separately clicked with heavy-isotope biotin-alkyne or light-isotope biotin-alkyne, respectively. These two types of biotin-alkyne yield the same click reaction efficiency and do not produce biases, as tested in previous work. Then equal amounts of VE and DK click reaction were mixed, precipitated with methanol-chloroform method, where methanol, chloroform and ddH2O were sequentially added to sample with volume of sample: methanol: chloroform: ddH2O = 1:3:1:3. The mixture was vortexed and centrifuged at 15,000 rcf. Protein precipitates were then resuspended with 8M Urea (MilliporeSigma, Cat#51456) dissolved in 100mM Tris-HCl (pH = 8.5). Resuspension was then reduced and alkylated and diluted 4x with 100mM Tris-HCl (pH = 8.5). Then TrypZean (MilliporeSigma, Cat#T3568–10mg) was added to protein sample at a 1:20 ratio and incubated on a thermomixer at 37°C. The digested sample was centrifuged at 15,000 rcf for 10 min at RT. The supernatant was removed and incubated with 200μL DPBS-washed Neutravidin beads (ThermoFisher, Cat#29200) on a thermomixer for 2h at 22°C. Stringent wash steps were followed as described in the published protocol, with an extra 2M Urea wash right after affinity binding. Bound peptides were eluted with acidic elution buffer (800μL acetonitrile, 197μL UltraPure H₂O, 2μL formic acid and 1μL trifluoroacetic acid). Eluate was then vacuum-dried with SpeedVac (ThermoFisher, Model#DNA120–115) until all solvent evaporated.

Dried peptides were then resuspended in buffer A (5% acetonitrile, 95% UltraPure H2O and 0.1% formic acid), then analyzed on a Fusion Lumos Orbitrap tribrid mass spectrometer (ThermoFisher). The digest was injected directly onto a 25 cm, 100 μm ID column packed with BEH 1.7 μm C18 resin (Waters). Samples were separated at a flow rate of 400 nL/min on a nLC 1000 (ThermoFisher). Buffer A and B were 0.1% formic acid in 5% and 80% acetonitrile, respectively. A gradient of 1–25% buffer B over 120 min, an increase to 40% buffer B over 40 min, an increase to 90% buffer B over 10 min and held at 90% buffer B for a final 10 min was used for 180 min total run time. The column was re-equilibrated with 20 μL of buffer A prior to the injection of sample. Peptides were eluted directly from the tip of the column and nano-sprayed directly into the mass spectrometer, by application of 2.5 kV voltage at the back of the column. The mass spectrometer was operated in a data-dependent mode. Full MS scans were collected in the Orbitrap at 120K resolution with a mass range of 400–1500 m/z and an AGC target of 4e5. The cycle time was set to 3s, and within this 3s the most abundant ions per scan were selected for CID MS/MS in the ion trap with an AGC target of 2e4 and minimum intensity of 5000. Maximum fill times were set to 50 ms and 100 ms for MS and MS/MS scans respectively. Quadrupole isolation at 1.6 m/z was used, monoisotopic precursor selection was enabled, and dynamic exclusion was used with exclusion duration of 5 s. Proteomics data quantification and statistical analysis were performed on Integrated Proteomics Pipeline (IP2) (Bruker Scientific, http://www.bruker.com).

Nascent proteomic analysis with DiDBiT in cultured neurons

AHA-labeled cultured neurons were extensively washed with 1x DPBS before lysed in dish with 10% RIPA buffer (diluted in 1x DPBS to reduce the amount of detergent so not to interfere with click chemistry) and 1x protease inhibitor. A cell scraper was used to maximize the sample recovery. Protein concentration was determined with the DC protein assay, and protein samples treated under the same condition were pooled to get 2mg protein starting material for subsequent preparation of one biological replicate. Sample preparation and LC-MS/MS protocol are the same as in vivo ANL-labeled samples.

FUNCAT in cultured neurons

FUNCAT protocols for in vitro and in vivo samples were adopted from previously published guidelines⁸⁴ with a few modifications. Specifically, cultured neurons in 96-well plates were transferred from the incubator to RT. From this point on, the plate was wrapped in foil to protect it from light. Cultures were fixed with FUNCAT fix buffer (1mM MgCl2 (Sigma-Aldrich, Cat#M8266), 0.1mM CaCl2 (Sigma-Aldrich, Cat#C3881), 4% PFA, 4% sucrose (Sigma-Aldrich, Cat#G5400) in 1x PBS) for 20 min at RT, then permeabilized with 0.5% DPBS-Tx100 for 15 min at RT, followed by blocking with 0.1% Triton X-100 in B-Block (10% normal horse serum (Jackson Immuno Research Labs, Cat#008–000-121), 5% sucrose and 2% BSA in 1x PBS, pH = 7.4) for 1h at RT. Cultures were then washed with 0.1% DPBS-Tx100 for 15 min at RT. After washing, cultures were incubated in FUNCAT mix O.N. at RT, which was prepared with 2.4μL 85mM TBTA, 50mM TCEP, 2μL 2mM fluor-alkyne (AlexaFluor647-Alkyne (ThermoFisher, Cat#A10278) or AlexaFluor488-Alkyne (ThermoFisher, Cat#A10267)) and 4μL 50mM CuSO₄ mixed in 1mL fresh 1x DPBS. FUNCAT mix was filtered with a 5mL syringe and 0.22 μm syringe filter (MilliporeSigma, Cat#SLGPR33RS) to remove precipitates, if any. In each 96-well, enough FUNCAT mix was added to fill up the entire well, and the plate was sealed off with adhesive plastic film (Bio-Rad, Cat#MSB1001), and incubated inverted while covered with foil. This prevented adhesion of click reagent aggregates and significantly reduced background aggregates to almost none in our experience. The next day, the film was peeled off and the culture was further washed with 0.1% DPBS-Tx100 for 10 min at RT and the regular ICC protocol was used to label necessary markers before being imaged.

Pentylenetetrazol, bicuculline, actinomycin-D and anisomycin treatments

In adult mice, 50 mg/kg PTZ (dissolved in 1x PBS) or PBS control was i.p. injected to induce seizure. PTZ solution was made fresh before use. Mice were monitored for 30 min after injection to rate seizure level according to the Racine-Pinel-Rovner seizure behavioral scale (Table S6). Only mice reaching stage 6 or 7 were included in the following analysis. Stage 8 usually quickly transitioned to death, therefore was not included in this study. In total, about 80% of mice injected with PTZ fit the criteria and were included in this study.

For primary neuronal cultures, 10mM bicuculline (Bicuculline Methiodine, Tocris, Cat# 2503), 200x stock solution was prepared by dissolving powder into ddH₂O. The stock solution was stored as aliquots in −20°C. When treating neuronal cultures, an appropriate amount of 200x bicuculline stock was mixed with pre-warmed culture media and then added to each well, to reach final concentration of 50 μM in culture. ddH₂O was used as control for this experiment.

For primary neuronal cultures, 2.5mM actinomycin-D (Sigma Aldrich, Cat#A1410) 100x stock solution and anisomycin (Tocris, Cat#22862–76-6) 1250x stock solution were prepared by dissolving into DMSO (Fisher Scientific, Cat#BP231–100). Stock solutions were stored as aliquots in −20°C. When treating neuronal cultures, appropriate amount of stock solution was mixed with pre-warmed culture media and then added to each well, to reach final concentration of 25μM of actinomycin-D or 40μM of anisomycin in culture. DMSO was used as control for this experiment.

Reverse transcription qPCR

To perform RT-qPCR on cultured neurons, culture media was removed, and cells were collected with TRIzol reagent (Invitrogen, Cat#15596026), with a cell scraper. RNA microprep kit (Zymo Research, Cat#R1050) was used to extract total RNA from the neuron sample in each well, following the protocol specified by the kit. RNA concentration was measured with nanodrop, and the same amount of RNA was taken from each sample for reverse transcription. cDNA was synthesized using the SuperScript IV reverse transcriptase kit (Invitrogen, Cat#18090010) with random hexamers. qPCR was performed with 2 ng/μL cDNA as template, SYBR green master mix (Bio-Rad, Cat#1725121), primers for genes listed below and the Bio-Rad CFX384 qPCR system (Bio-Rad, Cat#1855484). Primers for immediate-early genes were obtained from previously published sequences.⁸⁵Others were provided by IDT PrimerTime qPCR primer assays services.

In situ BONCAT-PLA in vitro

PLA was performed to detect biotinylated newly-synthesized Emerin. Specifically, in situ BONCAT was performed following the in vitro FUNCAT protocol, substituting the 2μL 2mM fluor-alkyne with 0.8μL 5mM biotin-alkyne in the FUNCAT mix. The same inverted incubation method was also applied here. After in situ BONCAT, PLA was immediately performed using Duolink Proximity Ligation Assay kit (Sigma Aldrich) according to the manufacturer’s protocol. Briefly, cultures were blocked with ICC blocking buffer, and then incubated with primary antibodies, goat-anti-biotin and rabbit-anti-Emerin, diluted in ICC blocking buffer, following the ICC protocol of these two steps. Then, the culture was washed with PLA wash buffer A twice for 5 min at RT, then incubated with PLA probe diluted 1:5 in PLA antibody diluent (Anti-Goat-PLUS (DUO92003) and Anti-Rabbit-MINUS (DUO92005) probes) for 1h in a 37°C humidified oven. Following probe incubation, the culture was rinsed with PLA buffer A twice for 5 min at RT. Ligation and amplification with far-red detection reagent (DUO92013) were then performed strictly following the manufacturer’s protocol. After the final rinse with 0.01x PLA wash buffer B, the culture was quickly fixed with 4% PFA in PBS for 15 min at RT to crosslink and secure the PLA complex, and to quickly denature all the reagents used prior to this step. Then, the culture was further subjected to regular ICC protocol to label necessary markers before being imaged.

Molecular cloning

All plasmids used in this study have an AAV compatible backbone and were subsequently packaged into AAV. A template plasmid AAV-U6-MCS1-hSyn-FLEx-MCS2-mRuby-MCS3 was generated to build shRNA and fusion protein overexpression vectors. Digestion of the multiple cloning site (MCS) was performed using restriction enzyme from NEB Bio-Lab. Three shRNA sequences specifically targeting mouse Emerin mRNA were chosen from The Broad Institute RNAi Consortium shRNA library, with 1 against 3′UTR (TRCN0000240585) and 2 against CDS (TRCN0000216203 and TRCN0000240584). A scrambled shRNA sequence was used as control shRNA. DNA oligoes were designed to have sticky ends compatible with MCS1, annealed, phosphorylated and inserted into template vector through T4 ligation (NEB Bio-Lab, Cat#M0202M). For the Emerin-mRuby fusion protein overexpression vector, the Emerin coding sequence was PCR amplified from a cDNA library reverse-transcribed from mouse primary neuronal culture RNA extract (a gift from the Maximov lab). PCR primers were designed according to the mRNA sequence from NCBI Reference Sequence Database (NM_007927.3). Amplified cDNA was validated through gel electrophoresis and sequencing, then purified and inserted into MCS2 or MCS3 of the template plasmid using the In-Fusion assembly mix (Takara, Cat#638948). The template plasmid was used directly as the overexpression control. Both C terminus and N terminus mRuby fusion plasmids were created this way. Ligation/assembly was used to transform NEB Stable competent E. coli (NEB Bio-Lab, Cat#C3040H), spread on carbenicillin (Gibco, Cat#10177012) LB-agar plates. Single colonies were picked, miniprepped and sequenced. Clones with the correct insertion were picked based on the sequencing data, and then subsequently tested with HEK cell transfection. Both C terminus and N terminus tagged fusion proteins showed ER and nucleus membrane localization, similar to the endogenous Emerin localization pattern stained with antibody in mRuby-only control transfected HEK cell. The C terminus fusion protein was expressed at a higher level than the N terminus fusion protein in HEK cells. Therefore, the C terminus fusion plasmid was used for AAV packaging and future experiments. shEmd triple-transfection in HEK cells overexpressing mouse Emerin showed efficient mouse Emerin knockdown, compared to sham transfected as well as shScramble transfected. AAV-CaMKIIα-Cre was modified from AAV-CaMKIIα-EGFP-Cre from Addgene (Addgene #105551) by removing the EGFP sequence. AAV-vGat-Cre was made by replacing the promotor in AAV-CaMKIIα-Cre with the vGat promotor from hU6-gRNA-vGat-dCas9-EGFP described before.⁸⁶ AAV-hSyn-FLEx-jGCaMP7s was obtained from Addgene (Addgene #104491).

AAV production

AAV packaging was performed as previously described.⁸⁷ First, plasmids to be packaged were maxi-prepped (Zymo Research, Cat#D4203) according to the protocol described by the kit. AAV-293 HEK cells (Agilent, Cat#240073) were used and cultured in 15cm culture dishes with 5% FBS-DMEM (ThermoFisher, Cat#11995065) supplemented with 1% NEAA (ThermoFisher, Cat#11140–050), 1% Sodium pyruvate (ThermoFisher, Cat#11360–070), 1% GlutaMax and 1% P/S. Briefly, HEK cells were triple-transfected with pHelper, pAAV.DJ and the expression vector using PEI (Polysciences, Cat#23966–1). Culture media was collected 72h and 120h after transfection. Cells are scrapped and collected at the 120h time point. Viral particles are precipitated from media with PEG. The media pellet and the cell pellet were digested separately using salt activated enzyme and then combined after completion. Ultrahigh-speed centrifugation was used to purify viral particles in a sucrose density gradient using Optiprep. The purified virus was then subjected to 4 rounds of DPBS buffer exchange in Amicon filter tubes and concentrated to the desired volume during the final buffer exchange. AAVs were titered using the qPCR protocol described in Challis et al., 2019. From 5 15cm culture dishes, we usually recovered virus with a titer around 10¹³ vg/ml in 400–600μL final volume. The virus was then aliquoted and stored in −80°C, or 4°C after thawing. For primary culture experiments, AAV-GCaMP7s and AAV-shRNA were added to primary culture at a dosage of 5×10⁷vg per 96 well, and cre-delivering AAVs were added at a dosage of 2.5×10⁷vg per 96 well. Emerin OE AAVs and the corresponding control were added at 1.25×10⁷vg per 96 well, with 1.25×10⁷vg cre-delivering AAVs. In other well plate formats, virus dosage was adjusted proportionally to the surface area. This dosage allowed sufficient fluorescent signal for imaging and good OE and KD level for Emerin in our experiments, while maintaining culture health. For stereotaxic injections, viruses were used at a concentration of 1×10¹²vg/ml.

Calcium imaging

For calcium imaging in vitro, DIV4 primary mouse neurons were transduced with AAV-hSyn-FLEx-GCaMP7s and either AAV-CaMKIIα-cre or AAV-vGat-Cre. AAVs reach steady expression 7 days after transduction. Neuronal cultures were imaged on DIV12. Calcium transients were imaged using the ImageExpress HT.ai confocal high-content imaging system (IXM, Molecular Devices). Glass bottom 96 well plates were placed in the imaging system with environmental control (37C, humidified gas constituted by 5% CO2, 21% O2 and rest with N2) throughout the entire imaging period. A 10x Plan Apo 0.45NA objective was used with a confocal 50μm slit setting. Laser power is set at 2% and each frame is exposed for 50ms, equal to 20Hz sampling rate. Each well was imaged sequentially in 4 non-overlapping FOV, with each FOV was imaged for 2min. The area imaged in total accounts for 25.45% of the well area. Single z-plane single-channel t-series.TIF files were analyzed using Suite2P.⁸⁸ Default settings were used for ROI registration and spike detection. ROI detection was manually inspected for each movie file as a quality control. For each movie file, the total deconvoluted spike counts and averaged spike amplitude were calculated for each ROI using Python and Numpy package. Data visualization and statistics were performed using Prism GraphPad.

Stereotaxic injection in visual cortex

To deliver AAV stereotaxically into the binocular region of adult mouse visual cortex, mice were anesthetized with 5% isoflurane inside an anesthesia induction chamber and then head-fixed on stereotaxic frame (Kopf instruments, model-942 with digital display) with 1.5–2% isoflurane constantly delivered through a nose cone throughout the procedure. 0.25 mg/ml Flunixin (supplied by TSRI department of animal resources) was i.p. injected at 10 mL/kg and 0.3 mg/ml buprenorphine (Covetrus, Cat#055175) was i.m. injected at 2 mL/kg prior to surgery procedures for analgesic purposes. The hair was removed, and the exposed skin was sterilized with Povidone-Iodine as well as 75% ethanol. A midline incision was made to expose the skull above the visual cortex. A NanoFil 10μL syringe (WPI, Cat#NANOFIL) with 33-gauge beveled NanoFil needle (WPI, Cat#NF33BV-2) was loaded with AAV, motorized with an electrical micropump (WPI, Cat#UMP3T-2) to control the amount of liquid dispensed and navigated to visual cortex through the digital coordinates display. bregma was used as set to (0, 0, 0) reference. Coordinates for the binocular region of primary visual cortex are AP = −3.3, ML = +/− 2.8, and the needle was advanced along the dorsal-ventral axis with 2 stops at DV = −1.1 and −1.5 for virus injection. A small opening was made in the skull with a microdrill (RWD, Cat#78001) above the site selected for injection. The syringe was advanced slowly to the first injection site. At each DV stop, 600nL of AAV was dispensed at 80 nL/min. The syringe remained at each injection site for another 10min to increase local AAV diffusion. After injection and retraction of the needle, the skin was sutured (Healthcare Products, Cat#ETHJ392H), and the closure was further reinforced with Vetbond. Mice continued to receive the same analgesics regime for 3 days and were monitored for health daily. Animals were used for experiments after 4–5 weeks.

Visually-evoked potential surgery and recording

For VEP recording experiments, we used the Data Acquisition and Analysis MP150 System (BIOPAC Systems, Inc.) to aid in implanting the electrode and for VEP recording. AAV was delivered through the stereotaxic injection protocol as described above. After AAV was injected, 2 slightly larger openings were drilled at coordinates AP = −3.5, ML = +/− 3.2 to implant the recording electrodes bilaterally into the binocular regions of visual cortex, as described previously.¹⁰ 2 additional openings were drilled at coordinates AP = +1, ML = +/−1 for bilaterally implanting reference electrodes in the prefrontal cortex. The electrodes consist of an insulated stainless-steel wire (0.005″ diameter, 3 mm length, 005SW-30S, PlasticsOne Inc.). The insulation was burned to remove 2 mm of coating from one end of the wire and then it was inserted into a male pin (A-M systems, Protech International Inc) and crimped (AFM8 crimping tool, Bloomfield Hills, Mich). The insulation at the other end of the wire was burned to remove 0.5 mm of coating with the help of a magnifying glass and a ruler. The male pin was connected to a female miniature pin (0.032″×0.4″0.005″ diameter, 3 mm length, 005SW-30S, PlasticsOne Inc.), crimped to a standard touchproof connector to interfacing to 100C-series Biopotential amplifiers (Biopac Inc). The average active electrode impedance was 5.2 ± 2.42 kΩ (mean ± SD, Grass Electrode Impedance Meter, Model EZM3A, Quincy, MA).

Two reference electrodes were implanted first, slowly inserted 0.25mm under the dura, and secured in place with dental cement (Stoeling, Cat#100229–622). Two recording electrodes were implanted next, one by one. During the implantation, the recording electrode was held on a stereotaxic arm and connected to the MP150 amplifier system with real-time recording with AcqKnowledge data acquisition software. The corresponding reference electrode was connected during this process to test the circuit. As the recording electrode was lowered into the brain, the noise level significantly dropped when it touched the dura, and from this point, the recording electrode was further advanced for 250μm to reach layer 2/3 of visual cortex. The recording electrodes were then cemented to the skull with dental cement. Then, a 3-pronged head bar (design kindly shared by the Komiyama lab, UCSD) was cemented to the skull, providing head restraint during the VEP recording with a mini-clamp designed to fit the 3-pronged head bar. Mice were post-operatively cared for in the same way as the stereotaxic injection protocol.

4 weeks after implanting the electrodes, VEP recording was performed under dim red light at 4–6p.m. period with the mice dark-adapted for 1h prior to each session. Mice were anesthetized with 10 mg/ml xylazine (AnaSed, Cat#59399–110-20) i.p. injected at 3 mL/kg and 10 mg/ml pentobarbital (Pisa agropecuaria, PISAPENTAL) i.m. injected at 3 mL/kg. Mice were placed in the head-restraint mini-clamp and positioned with their eyes 20cm in front of a LED stroboscope (Monarch instruments, Nova-pro 300) controlled by the MP150 system to flash at 1Hz of 0.05 cd s/m² white light. 250 flashes were delivered binocularly while simultaneously recording visually evoked response from both hemispheres. The signal was amplified 10,000 (PM150 system, Biopac Inc), bandpass filtered (1–35 Hz) and digitized at 20000 Hz with 12 bits resolution. Responses were averaged across 250 iterations within 1 session. 2 sets of 2 amplifiers were used to measure each hemisphere and then switched between the 2 hemispheres and recorded again to exclude potential instrument bias. 4 traces were obtained for each visual cortex response and averaged to quantify response amplitude and latency.

Visual cliff test and video analysis

The visual cliff apparatus is a home-made acrylic plexiglass box as depicted in Figure 7, placed inside a dark room with dim red light. The top part is a 60 cm × 60 cm × 60cm plexiglass box enclosing a flat transparent floor with black and white checkerboard pattern at 100% contrast attached to the underside of one quadrant of the floor (the safe zone). White paper was taped around the outside to block irrelevant visual cues. The bottom part of the apparatus is 60 cm × 60 cm × 50cm in size, with the same black-white checkerboard pattern covering the 4 walls and the floor. A Logitech Brio 4K webcam was held right over the top of the arena walls recording at 30 frames per second. A light source was placed next to the webcam to provide lighting for the test area.

With this setup, the texture of the safe zone and the remainder of the transparent floor on the top part is homogeneous to the mice, and the proximal visual cues inside the safe zone can be distinguished from the distant visual cues on the lower floor of the unsafe zones. Our visual cliff test, slightly different from the conventional visual cliff setup, is constituted by 25% of safe zone and 75% unsafe zone, as opposed to the traditional 50–50 division. This setup doubles the chances of crossing the edge from safe to unsafe zones (2 edges as opposed to 1 edge in 50–50 division), which seemed to help animals recognize the differences between the safe and unsafe zones sooner and decide to stay in the safe zone faster if their depth perception was intact. All visual cliff tests were performed at 1–3p.m. Prior to tests, mice were acclimated in the testing room for 1h. During the test, a mouse was placed in the center of the safe zone, and then allowed to explore freely for a 2min period. Apparent safe zone preference was detected within this 2min period, and this preference didn’t change when tested a second time 4 weeks later. The plexiglass surface of the top part (the walls and the floor) was thoroughly cleaned with disinfecting and deodorizing spray (PeroxiGard, AHP, Cat#74559–4) in between trials. Videos were post-processed with Capcut movie processing software to remove the frames involving the experimenters. Then, mice movements and arena landmarks were labeled and traced using DeepLabCut Resnet50 machine learning network following previously published guidance.⁸⁹ Python-based DLCAnalyzer was modified and applied to define safe/unsafe zones, calculate speed/time/distance and generate zone visit reports.⁹⁰

IXM high-content imaging, image processing and analysis

Neuronal cultures in glass-bottom 96 well plates (processed according to the ICC, FUNCAT or in situ BONCAT-PLA protocols) were imaged using the HT.ai confocal high-content imaging system (Molecular Devices). The imaging was set up using either the 20x Apo LWD (NA 0.95) or the 40x Apo LWD (NA 1.15) water immersion objectives, with 4 available channels including DAPI, FITC, TRITC and Cy5. 16–36 non-overlapping FOV were imaged per well. All datasets were manually inspected for out-of-focus images or images including well edges as quality control. Images were pre-processed using ImageJ in a batch mode before Cell Profiler analysis.⁹¹ Then, customized Cell Profiler pipelines were written and applied to each dataset based on the experimental designs. In general, a pipeline includes illumination correction, primary ROI identification with threshold and watershed, relating objects, image/object-based measurements, ROI overlay output, and data exportation in database format. Databased files were further read by DB browser software and per-image and/or per-object information was exported to.csv format for statistics.

In greater detail: The images were acquired on an HT.ai confocal high-content imaging system (Molecular Devices) under the full 16-bit range with the use of the saturation assistance to avoid saturated pixels. Image data were analyzed using ImageJ as described below: (1) To analyze cell body signals in Figures 2D, 2H, 3F, 4D–4F and 5SJ, the DAPI channel was thresholded, and size-filtered to generate a mask for individual neuron measurements. The channel that we were quantifying (Emerin, BONCAT-PLA, FUNCAT or EGFP signals) was not subjected to post-imaging processing. (2) To analyze puncta signals in Figures 5 and S6, the DAPI channel was thresholded, watershed and size-filtered to count neurons in the field-of-view and the puncta signals (synaptic makers) are thresholded to remove background, watershed and size-filtered to facilitate puncta counting. Parameters for each post-processing steps were chosen and tested on 20 images per condition to ensure sufficient background removal and avoid signal loss/saturation. Once chosen, the same set of parameters were applied to all images in the same channel of different conditions in the entire dataset. (3) To analyze full field signals in Figures 6I and S8A, images were not subjected to post-imaging processing, however representative images were further processed for demonstration purposes. The min and max intensities were adjusted to the full dynamic range, and the same min/max values were applied to all images in the same channel of that dataset.

We established an automated method to identify and count neurons or dead cells with condensed nuclei, using DAPI labeling and size filters. To count neurons, we used MAP2 immunolabeling as a reference to evaluate the performance of DAPI-based cell identification and counting. A stringent minimum and maximum size range for the DAPI labeled nuclei allows exclusive identification of MAP2+ cells (i.e., neurons) and excludes dead cells, with small bright condensed nuclei. This strategy minimized manual or investigator bias and generated more consistent data across samples. To quantify immunocytochemistry signals in neuronal cell bodies, each neuronal cell body, determined using size-filtered expanded DAPI object, was used as the ROIs to measure signal intensity in the channel-of-interest. The size threshold was chosen empirically to exclusively retain neuronal nuclei and manually validated using MAP2 immunolabeling. To count fluorescently labeled puncta, intensity-based filters were chosen and evaluated manually to ensure sufficient background removal and minimize signal loss and were applied to all images in the same channel across different conditions. Puncta were segmented using watershed and counted. Neurons were identified as size-filtered DAPI objects, counted and used to normalize puncta count to determine puncta density per neuron. While allowing synaptic marker density comparison between treatment and control, synaptic density counts underestimate the true synapse numbers in our cultures, based on spine density as shown in Figures 5I–5K (avg. 0.2 spine/μm).

Nikon confocal imaging and analysis

Mouse brain sections were imaged on a Nikon A1 confocal microscope with either the 10× Plan Apo objective (NA 0.45), the 20× Plan Apo objective (NA 0.7) or the 40× Plan Fluor oil immersion objective (NA 1.3). cFos expression was analyzed through manual cell counting assisted with ImageJ cell counter plugin.

Bioinformatics

Bioinformatic analyses were performed using the QIAGEN IPA (QIAGEN Inc.),⁹² Metascape database⁹³ and STRING PPI network analysis.⁹⁴ For IPA, core analysis was performed using p-value (0.05 cutoff) and log₂ expression ratio as data input. The same datasets were submitted to Metascape for parallel analysis. Pathway components identified by Metascape were then input to the STRING database to generate PPI networks with orphan nodes removed. For the IPA analysis shown in Figure 1E, p values are indicated by the size of each dot in this plot, while colors indicate the ‘IPA Activation Z score’. The enrichment p value of each pathway only plays a partial role in calculating the activation Z score by the IPA software. This value considers two main factors: (1) the direction of regulation, ‘up’ or ‘down’, of the genes/proteins in this pathway according to the user-submitted datasets and (2) the literature-derived direction of the gene-to-pathway effect, which is either ‘increasing’ or ‘decreasing’. When clear literature data are not available on the directionality of how a gene/protein regulates a pathway, the subsequent activation prediction will return ‘not available’ (gray). This doesn’t invalidate this pathway’s enrichment, and therefore it’s included.

Statistics

Statistic tests were selected according to the experimental design and types of data and performed using Prism GraphPad unless otherwise stated, as indicated in the figure legends. Normally distributed datasets were analyzed used parametric statistical tests while non-normal datsets were analyzed using non-parametric tests with adjustments for multiple comparisons as necessary. The following tests and corrections were used: two-tailed t tests, Welsh’s t test, paired t test, Pearson correlation coefficient (R), one-way ANOVA with Sidak correction for multiple comparisons, Kruskal-Wallis test with Dunn’s correction for multiple comparisons, Mann-Whitney test. Proteomics data quantification and statistical analysis were performed on Integrated Proteomics Pipeline (IP2) (Bruker Scientific, http://www.bruker.com). The statistical significance of the differential expression of all proteins was determined using a two-tailed t test of the corresponding peptide quantification ratios between the two conditions with p < 0.05 set as the cutoff for statistical significance. False discovery rate (FDR)-adjusted p values were calculated using the Benjamini-Hochberg correction. The statistical tests, plot types, N values, and data representation are specified in the figure legends. In some plots, confidence intervals or error bars are obscured by the median or the mean. Normalization was noted in the y axis label or figure legends. Data were analyzed blind to experimental condition.