Primary cell cultures of rat cerebellar granule cells

For each isolation of cells, mixed-sex litters of 10 Wistar 8-day-old rat pups were obtained. The pups were euthanized by decapitation on the day of experiment, without prior use of anaesthesia. Animals were kept in standard cages under a 12-h light–dark schedule at a constant temperature of 21 °C with food and water ad libitum, in an authorized facility at Santa Lucia Foundation, Rome (Italy). All procedures involving rats complied with the Istituto Superiore di Sanità (Italian Ministry of Health) and current European Ethical Committee guidelines (directive 86/609/ECC). Efforts were made to minimize animal suffering and to reduce the number of animals used. Primary cultures enriched in post-mitotic cerebellar granule cells (GCs) were obtained by dissociation of cerebella from 8-day-old rat pups, following the procedure described by Levi et al. (1984). Cells were seeded (3 × 106 cells/dish) in 35-mm plastic dishes previously coated with poly-L-lysine using Gibco Basal Medium Eagle (BME, ThermoFisher Scientific, Walthman, MA, USA) supplemented with 10% heat-inactivated foetal calf serum (Gibco, ThermoFisher Scientific), 2 mM glutamine (Gibco, ThermoFisher Scientific), and 25 mM KCl and 100 µg/ml gentamycin (Gibco, ThermoFisher Scientific). To ensure a good purity of cerebellar granule cells 24 h after cell plating, we added Ara-C (10 mM; Sigma Aldrich, St. Louis, MO, USA) to the culture medium to prevent proliferation of non-neuronal cells. This protocol typically yields cultures of cerebellar granules that are highly enriched (> 90%) and extremely pure since Ara-C functions as anti-mitotic agent by inhibiting DNA synthesis in dividing cells (Drejer and Schousboe 1989). This mechanism ensures that all non-neuronal, dividing cells, potentially present in our cultures, are negatively selected and washed away by protecting neuronal cells that are less proliferating.

SH-SY5Y cell cultures

In this study, we also used two SH-SY5Y clones, well characterized in previous papers (Maresca et al. 2012; Nardella et al. 2015), derived from the parental SH-SY5Y human neuroblastoma cell line (ATCC), LMNA-knockdown (KD) and Mock cells that were obtained and cultured as previously described (Maresca et al. 2012). Cells were cultured in a 1:1 mixture of Eagle’s Minimum Essential Medium and F12 medium (Gibco, ThermoFisher Scientific) supplemented with 10% Fetal Bovine Serum (FBS; Hyclone, ThermoFisher Scientific), 2 mM L-glutamine, 0.5% non-essential amino acids, 0.5% sodium pyruvate and 1% penicillin and streptomycin, in the presence of blasticidin.

Lentiviral infection for generation of Lmna-KD rat cerebellar GCs

GCs dissociated from rat cerebella were seeded (3.5 × 105 cells/dish) in 35-mm plastic dishes previously coated with poly-L-lysine. After four hours, they were exposed to the virus-containing supernatant from pLenti6/V5-GW-miR-LMNA or pLenti6/V5-GW-miRNeg (Maresca et al. 2012). After 18 h, fresh medium was added. The miRNA expression was monitored by checking the  Lmna gene expression.

Lmna knock-out (KO) mice

The generation of Lamin Δ8–11 knock-out mouse (Lmna − / −) used in this study has been already described (Sullivan et al 1999; Jahn et al. 2012; Cesarini et al. 2015). The animals were kept, as described above, in an authorised facility at Santa Lucia Foundation, Rome (Italy). Cerebella of 8-day-old wild type (n = 8) and Lmna-KO (n = 9) mice were dissociated, and cultures enriched in granule neurons were obtained according to the procedure described by Levi et al. (1984) as above described.

Glutamate excitotoxicity

The evaluation of glutamate excitotoxic effect was performed in all the three models employed using multiple cell viability assays.

For rat cerebellar GCs, after 2, 5 and 8 days in culture, they were washed once in Locke solution (in mM: 154 NaCl, 5.6 KCl, 3.6 NaHCO3, 2.3 CaC12, 1.0 MgC12, 5.6 glucose, 10 HEPES, pH 7.4) and exposed to a 100 µM glutamate pulse in Mg2+ -free Locke solution for 30 min at room temperature. Cells were subsequently washed in Mg2+ -free Locke solution and returned to the incubator in their original medium. After 18 h, counting of the numbers of intact nuclei was used to determine the number of viable cells, as reported by Soto and Sonnenchein (1985) and modified for counting cerebellar granule cells following the procedure described by Volonté et al. (1994).

The same assay was performed in Lmna-KO mouse cerebellar GCs after 8 days in culture. The cells were treated as described above for rat GCs.

For human SH-SY5Y cells, Mock and LMNA-KD cell cultures were exposed to different concentrations of glutamate (0.1, 10 and 60 mM) for 24 h. Then, cells were harvested, washed once in PBS and analysed for viability by using propidium iodide (PI) and FACS. Cells were suspended in a solution of PBS containing 10 µg/ml PI and incubated for 1 min at room temperature in the dark, then directly measured by a FACSCalibur cytometer (Becton Dickinson) and CellQuest Pro BD software (Becton Dickinson). Flow cytometry data were analyzed by FlowJoTM data analysis platform v.8.0 (Becton Dickinson). PI cell viability assay was also used to evaluate cell viability in SH-SY5Y Mock and LMNA-KD cells in the presence of the Ca2+ chelator 1,2-bis(o-Aminophenoxy)ethane-N,N,Nʹ,Nʹ-tetraacetic Acid Tetra(acetoxymethyl)Ester (1.5 mM; BAPTA-AM, Molecular Probes). Cells were pre-incubated 20 min with the chelator, which was maintained during the treatment in the presence of glutamate 60 mM for 24 h. The PI assay was performed as described above and samples analyzed by FACS. The presence of viable cells after exposure of Mock and LMNA-KD SH-SY5Y to glutamate 60 mM in absence or presence of the Ca2++ chelator was also evaluated by the Cell Counting Kit-8 (CCK8) assay (Merck KGaA, Darmstadt, Germany). 96-well plate cell cultures were exposed to glutamate 60 mM in the absence or presence of the Ca2+ chelator as described above. After 24 h the CCK-8 solution was added to each sample following the manufacturer’s protocol for 3 h at 37 °C. Absorbance was then measured at 450 nm using the Varioskan™ LUX Multimode Microplate Reader (Thermofisher Scientific, Waltham, MA, USA).

Annexin V assay

Mock and Lmna-KD GCs were treated with 100 µM glutamate as described above. They were then harvested, pooled with the supernatant, washed once in PBS and processed for Annexin V assay. Vybrant Apoptosis assay kit (Invitrogen) was used following the manufacturer’s protocol and samples analyzed by FACSCalibur cytometer (Becton Dickinson) and CellQuest Pro BD software (Becton Dickinson). Flow cytometry data were analyzed by FlowJo™ data analysis platform v.8.0 (Becton Dickinson).

Confocal Immunofluorescence of cerebellar tissue

Confocal analysis of Lamin A/C in foetal (E10) and neonatal (P10 and P18) rat cerebella were performed. After perfusion with saline, under deep anesthesia (60 mg/kg Nembutal i.p.), followed by cold 4% PFA in 0.1 M Phosphate Buffer, pH 7.4, the brains were excised, cerebella isolated and cryoprotected in 30% sucrose/PB at 4° C. They were then frozen with dry ice and cut into 40-μm transverse sections with a freezing microtome. Non-specific staining was blocked by incubating sections in blocking buffer (0.25% Triton X-100, 5% normal donkey serum in PBS) for 1 h at room temperature. Antibodies used: mouse monoclonal antibody anti-NeuN (A60, Sigma Aldrich) and goat polyclonal anti-Lamin A/C (N18, Santa Cruz Biotechnology, TX, USA). All antibodies were diluted in PBS containing 5% normal donkey serum and 0.01% Triton X-100. After overnight incubation and three washes in PBS, primary antibody staining was revealed using fluorescence-conjugated secondary antibodies (Thermo Fisher Scientific) at 2 µg/mL. Sections were washed three times in PBS, mounted on gelatin-coated slides, and coverslipped in ProLong Gold Antifade Reagent (Thermofisher Scientific). Nuclei were stained with DAPI (P36935, Invitrogen, Carlsbad, CA, USA). For the negative control, sections were treated following the previously outlined protocol, except for omitting the primary antibody. Sections were examined under a confocal laser scanning microscope (LeicaSP5, Leica Microsystems, Germany) under sequential mode to avoid crosstalk between channels. Image processing and final figures were done by using AdobePhotoshop 7 and Adobe Illustrator 10. Fluorescence intensity measurement was performed by ImageJ software. The Integrated Density value (the product of Area and Mean Gray Value) was calculated for each image (n = 3) of nuclei, NeuN and Lamin A/C. Quantification was performed by calculating the Ratio between the NeuN or Lamin A/C fluorescence and the nuclei fluorescence.

Immunofluorescence of primary rat GCs

Rat GC neuronal cultures were washed twice with PBS and fixed in 4% (w/v) paraformaldehyde for 15 min at room temperature. Cells were permeabilized with 0.1% (v/v) Triton X-100/PBS, pH 7.4, for 4 min at room temperature. Coverslips were saturated with 2% BSA and 10% normal goat serum (NGS) for 3 h followed by incubation overnight at 4 °C in a humidified chamber with the mouse monoclonal antibody anti-NeuN (A60, Sigma Aldrich) and goat polyclonal anti-Lamin A/C (N18, Santa Cruz Biotechnology, TX, USA). Unbound antibody was removed by three washes with PBS and bound antibodies were detected by incubation with donkey anti-mouse Alexa 488 (Thermo Fisher Scientific) and donkey anti-goat Alexa 594 (Thermo Fisher Scientific) secondary antibodies at room temperature for 30 min. Nuclei were stained with nuclear dye 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma, St. Louis, MO, USA) 1:1000 in PBS for 5 min. Controls were performed by omitting the primary antibody. Then, coverslips were mounted on Superfrost glass slides using the Prolong Gold Antifade Mounting (Thermo Fisher Scientific) and kept at −20◦C before image analysis. Immunofluorescence images were acquired with an epifluorescent microscope (Leica CTR5500; Leica Microsystems, Mannheim, Germany) equipped with a CCD camera (Leica). Final figures were assembled by using Adobe Photoshop 7 and Adobe Illustrator 10. Images are representative of at least three independent experiments. Fluorescence intensity measurement was performed by ImageJ software. The Integrated Density value (the product of Area and Mean Gray Value) was calculated for each image (n = 3) of nuclei, NeuN and Lamin A/C. Quantification was performed by calculating the Ratio between the NeuN or Lamin A/C fluorescence and the nuclei fluorescence.

Western blotting

Cultured cells were washed twice with 1X PBS and then incubated for 1 min in 1X PBS added with 0.5 mM phenylmethylsulphonyl fluoride (PMSF; Sigma-Aldrich) and 1X Complete Protease Inhibitors (Sigma), then scraped, harvested, and briefly sonicated. Proteins were suspended in urea buffer (8 M urea, 100 mM NaH2PO4, and 10 mMTris pH 8) and the protein concentration was calculated with Bradford assay (Bio-Rad Laboratories S.r.l., Segrate, Italy). Thirty µg of proteins were subjected to SDS–polyacrylamide gel electrophoresis with NuPAGE kit (Life Technologies, Carlsbad, CA, USA) according to manufacturer's instructions. Resolved proteins were blotted overnight onto nitrocellulose membranes, which then were blocked in 1X PBS containing 5% non-fat milk for at least 1 h. Blots were incubated with the following primary antibodies: goat polyclonal anti-Lamin A/C (N-18; Santa Cruz Biotechnology, Dallas, TX, USA); mouse monoclonal anti-GAPDH (6C5; Merck Life Science, Milan, Italy). After four washes (10 min/each) in 1X PBS and 0.1% Tween20, the membranes were incubated for 45 min with the appropriate secondary antibody: donkey anti-goat IRdye800 (LI-COR Biosciences, Lincoln, NE, USA) or donkey anti-mouse IRdye800 (LI-COR Biosciences). The membranes were then analysed with the Licor Odyssey Infrared Image System (LI-COR Biosciences) in the 800 nm channel. Densitometry analyses were performed using ImageJ software.

Intracellular calcium analysis

Mock and Lmna-KD rat GCs were cultured in 96-well plates as described above. After 8 days in vitro (8DIV), GCs were washed once in Locke solution (in mM: 154 NaCl, 5.6 KCl, 3.6 NaHCO3, 2.3 CaC12, 1.0 MgC12, 5.6 glucose, 10 HEPES, pH 7.4) and exposed to a 100 µM glutamate pulse in Mg2+-free Locke solution for 30 min at room temperature. Cells were subsequently washed in Mg2+-free Locke solution and incubated with Ca2+ sensor Fluo-4 (Fluo-4 Direct Calcium Assay Kits, Molecular Probes, Eugene, OR, USA) for 40 min at 37 °C in medium containing probenecid 5 mM following manufacturer’s protocol. Green fluorescence (excitation at 494 nm and emission at 516 nm) was then measured using the Wallac 1420 Victor2 fluorescence microplate reader (Perkin Elmer, Shelton, Connecticut, USA).

Mock and LMNA-KD SH-SY5Y cells were cultured on Poly-D-Lysine coated glass culture slides at a confluence of about 70%. Cells were then incubated with Ca2+ sensor Fluo-4 (Fluo-4 Direct Calcium Assay Kits, Molecular Probes) for 40 min at 37 °C in medium containing probenecid 5 mM following manufacturer’s protocol. Cells on the slide were then transferred to the stage of an upright fluorescence microscope (Bx51, Olympus, Tokyo, Japan) and continually superfused with an extracellular medium (Locke solution, see above). After 90 s, glutamate was superfused, within the same extracellular medium, at 60 mM concentration (IC50). Fluo-4 was excited by light at 490 nm from collimated LED (Thorlabs, Newton, NJ, USA), for 100 ms every cycle of image acquisition (1 s). LED emission was filtered through interference band–pass filters centered on peak wavelength, attenuated with a neutral density filter and conveyed onto the sample by reflection off a DM480HQ dichromatic mirror (Olympus). Fluorescence emission was collected through an interference filter (BA495–540HQ, Olympus) using a water immersion objective (20x, N.A. 0.95, XLumPlanFI, Olympus). Images were formed on a CCD camera (PCO Sensicam, 100 ms exposure time/frame) controlled by software developed in the laboratory. Images were analyzed with software developed in the laboratory using the MATLAB platform (Release 14, MathWorks, Inc., Natick, MA, USA). Fluo-4 traces were generated by averaging pixel signals within regions of interest (ROIs) corresponding to individual cells.

Intracellular calcium was also analysed by flow cytometry. Mock and LMNA-KD SH-SY5Y cells were cultured in 35-mm Petri dishes at a confluence of about 70%. Cells were then exposed to Ca2+ sensor Fluo-4 (Fluo-4 Direct Calcium Assay Kits, Molecular Probes) for 40 min at 37 °C in medium containing probenecid 5 mM following manufacturer’s protocol. Cells were collected in trypsin/EDTA solution, centrifuged at 200 × g for 5 min and suspended in Locke solution. Glutamate 60 mM was then added to the cell suspension for 30 min at room temperature and the cell-associated fluorescence was measured by a FACSCalibur cytometer (Becton Dickinson) and CellQuest Pro BD software (Becton Dickinson).

RNA extraction

Total RNA was isolated from the different cell types using the Total RNA purification kit (Norgen Biotek, Thorold, ON, Canada), according to manufacturer’s protocol. Total RNA was quantified by Quant-it™ RiboGreen RNA Assay Kit (Invitrogen, ThermoFisher Scientific) according to the manufacturer’s instructions. The absorbance of the samples was measured by a multimode microplate reader (Varioskan Lux, Life Technologies) at a wavelength of 530 nm. RNA integrity was determined with a BioAnalyzer 2100 instrument, using a RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA). Samples with an RNA Integrity Number (RIN) index lower than 8.0 were discarded.

Microarray gene expression profiling of rat GCs

Gene expression profiling of Lmna-KD and Mock rat cerebellar GCs was performed using the Agilent Whole Rat Genome 4 × 44 K microarray platform (Agilent Technologies). Briefly, the cyanine 3-CTP labeled cRNA samples were prepared using the Agilent Low Input Linear Amplification Kit (Agilent Technologies). After hybridization and washing, array images were acquired using the Agilent Scanner G2564C (Agilent Technologies) and signals were extracted by Agilent Feature Extraction software (ver 10.7.3.1), according to the standard Agilent one-color gene expression extraction protocol (GE1_1100_Jul11). Data quality filtering and normalization were performed and differentially expressed genes (DEGs) in Lmna-KD vs. Mock cells were identified as those having a fold-change ratio |Lmna-KD/Mock|> 1.5 in linear scale. Probe annotation file was downloaded from NCBI GEO repository (Platform GPL7289, Last update date Apr 06, 2012). ClusterProfiler R package (v.3.18.1) (Yu et al. 2012) was used for the functional enrichment analysis of annotated DEGs and Gene Ontology (GO) Biological Process (BP) and Molecular Function (MF) categories were retrieved from the Rattus Norvegicus org.Rn.eg.db R package (v.3.12.0). The whole list of genes on the Agilent array (17,366 annotated genes) was used as background. A false-discovery-rate (FDR, Benjamini–Hochberg correction) threshold < 0.05 was applied to all the annotation terms to define statistically significant enrichments.

RT-qPCR

The expression of differentiation and stemness genes in rat GCs was evaluated by RT-qPCR at different days during culturing.

To validate the microarray data obtained in Lmna-KD rat GCs, we also performed in Lmna-KO mice the RT-qPCR of some selected genes whose expression was up- or down-regulated by Lmna silencing in rat cerebellar GCs in vitro. Total RNA was isolated from wild type (n = 4) and KO (n = 4) mice as above described. RNA was quantified by Quant-it™ RiboGreen RNA Assay Kit (Invitrogen) according to the manufacturers’ guidelines. RNA integrity was checked on 2200 TapeStation System (Agilent Technologies). Samples with a RIN lower than 8.0 were discarded.

For all samples, RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. Equal amounts of cDNA were then subjected to real-time qPCR analysis on QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems) with PowerUp™ SYBR™ Green Master Mix (Applied Biosystems).

The primers listed below (r for rat, m for mouse) were used at final concentration of 200 nM using Tbp or Ppia as endogenous controls: rTbp (For: 5’-CCCACCAGCAGTTCAGTAGC-3’, Rev: 5’-CAATTCTGGGTTTGATCATTCTG-3’); rLmna (For: 5’-GAGCAAAGTGCGTGAGGAGT-3’, Rev: 5’- TCCCCCTCCTTCTTGGTATT-3’); rProm1 (For: 5’-GCCCAAGCTGGAAGAATATG-3’, Rev: 5’- CAGCAGAAAGCAGACAATCAA-3’), rNes (For: 5’-TCCCTTAGTCTGGAAGTGGCTA-3’, Rev: 5’- GGTGTCTGCAAGCGAGAGTT-3’); rZic2 (For: 5’-TCAACATACCAACCCATAGC-3’, Rev: 5’-AAAAATACATTCACAAGCGTTGG-3’); rGabra6 (For: 5’-AATGTCAGTCGGATTCTTGACA-3’, Rev: 5’- TGTTTTGACTTCTGTTACAGCAC-3’); mPpia (For: 5’-CCCACCGTGTTCTTCGACAT −3’, Rev: 5’- CCAGTGCTCAGAGCTCGAAA-3’); mCcl5 (For: 5’-CCAATCTTGCAGTCGTGTTTGT-3’, Rev: 5’-CCCTCTATCCTAGCTCATCTCCA-3’); mCcl7 (For: 5’-TCACCAGTAGTCGGTGTCCC-3’, Rev: 5’-ACCCACTTCTGATGGGCTTC-3’); mCxcl10 (For: 5’-CGTGTTGAGATCATTGCCACG-3’, Rev: 5’-TGGTCTTAGATTCCGGATTCAGA-3’); mKcnma1 (For: 5’-CGTGGGTCTGTCCTTCCCTA-3’, Rev: 5’-TCCCAGGGTTAATTAATATTCGGCT-3’); mKcnmb2 (For: 5’-GCAGAGCGTGTGGACAGAAG-3’, Rev: 5’-GGCAAGGGTACTGAGAGAGC-3’); mKcnt1 (For: 5’-CCACCACTGGCTATGAGGAC-3’, Rev: 5’-AGGGTGTTCTGGTGATGATCG-3’).

Microarray gene expression profiling of SH-SY5Y human neuronal cells

Gene expression profiling of LMNA-KD and Mock SH-SY5Y neuroblastoma cells was performed using the Agilent one-color microarray standard protocol and was already deposited in GEO Series GSE30677 (Maresca et al. 2012). Briefly, the cyanine 3-CTP labeled cRNA samples were prepared using the Agilent Low Input Linear Amplification Kit (Agilent Technologies) and hybridized onto Agilent 4 × 44 K Whole Human Genome oligonucleotide microarray (GEO platform GPL6480). Images were acquired using the Agilent Scanner G2564B by Agilent Feature Extraction software (ver 10.1), using the one-color gene expression extraction protocol (GE1_107_Sep09). DEGs were defined as those having a fold-change ratio |LMNA-KD/Mock|> 1.5 in linear scale. Probe annotations were downloaded from the Agilent portal web (https://earray.chem.agilent.com, annotation version 08-Nov-2022).

Functional enrichment analysis of annotated DEGs was performed using ClusterProfiler R package (v.3.18.1) (Yu et al. 2012) and Gene Ontology (GO) Biological Process (BP) and Molecular Function (MF) categories retrieved from the Homo sapiens org.Hs.eg.dbR package (v.3.12.0). The whole list of genes on the Agilent array (14,554 annotated genes) was used as background. A false-discovery-rate (FDR, Benjamini–Hochberg correction) threshold < 0.05 was applied to all the annotation terms to define statistically significant enrichments.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5 software (GraphPad software, Inc., La Jolla, CA, USA). One-way ANOVA was used for groups of data. Unpaired student’s t test was used for comparison of pairs of data.