Impaired expression of the COSMOC/MOCOS gene unit in ASD patient stem cells

Autism spectrum disorders (ASD) are complex neurodevelopmental disorders with a very large number of risk loci detected in the genome. However, at best, each of them explains rare cases, the majority being idiopathic. Genomic data on ASD derive mostly from post-mortem brain analyses or cell lines derived from blood or patient-specific induced pluripotent stem cells (iPSCS). Therefore, the transcriptional and regulatory architecture of the nervous system, particularly during early developmental periods, remains highly incomplete. To access the critical disturbances that may have occurred during pregnancy or early childhood, we recently isolated stem cells from the nasal cavity of anesthetized patients diagnosed for ASD and compared them to stem cells from gender-matched control individuals without neuropsychiatric disorders. This allowed us to discover MOCOS, a non-mutated molybdenum cofactor sulfurase-coding gene that was under-expressed in the stem cells of most ASD patients of our cohort, disturbing redox homeostasis and synaptogenesis. We now report that a divergent transcription upstream of MOCOS generates an antisense long noncoding RNA, to which we coined the name COSMOC. Surprisingly, COSMOC is strongly under-expressed in all ASD patients of our cohort with the exception of a patient affected by Asperger syndrome. Knockdown studies indicate that loss of COSMOC reduces MOCOS expression, destabilizes lipid and energy metabolisms of stem cells, but also affects neuronal maturation and splicing of synaptic genes. Impaired expression of the COSMOC/MOCOS bidirectional unit might shed new lights on the origins of ASD that could be of importance for future translational studies.


ASD patients and Cell cultures
A complete written and oral information on the goal and procedure of this research was provided to the participants or their legal tutors and a signed informed consent was obtained from all of them before their involvement in the study. All procedures were approved by the local committee (Comité de Protection des Personnes, files#205016 and #205017) of Marseille.For ASD patients, nasal biopsies were performed by an ENT surgeon requiring a general anesthesia.
Human nasal olfactory stem cells (OSC) from 11 patients and 11 age-and gender-matched control individuals without diagnosed neuropsychiatric disorders were isolated and primary cultures were established as previously described (1). Cells were used at the same and low passage number and were regularly controlled as free of mycoplasma contamination throughout the study. Primary OSCs as well as SH-SY5Y, U138-MG, HepG2, HEK-293 and Caco-2 cell lines were cultured with DMEM high glucose media containing Glutamax media (Life Technologies) and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. THP-1 cells were cultured with RPMI media (Life Technologies), 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were incubated at 37°C in a humidified chamber containing 5% CO2. Culture media was changed every three days and cells were passaged with Trypsin/EDTA (Life Technologies) when they reached ~90% confluency.
For induced pluripotent stem cells (iPSCs) culture, OSCs from two control individuals were reprogrammed by using a retroviral-based reprogramming strategy. In short, cells were infected with an equal ratio of retroviruses encoding for Oct3/4, SOX2, Klf4 and c-MYC by spinfection of the cells at 1,850 rpm for 1 hour at 32°C in the presence of polybrene (4 mg/ml). After two serial infections, cells were passaged onto fresh irradiated mouse embryonic fibroblasts (iMEFs, amsbio) and switched to a medium containing DMEM/F12 (Life Technologies) supplemented with 20% Knockout Serum Replacement (Life Technologies), 1 mM L-glutamine (Life Technologies), 0.1 mM non-essential amino acids (Life Technologies), 55 mM bmercaptoethanol and 10 ng/ml bFGF (PeproTech). For the derivation of hiPSC lines, iPS-like colonies were manually picked and maintained on fresh iMEF feeder layers for four to five passages before being transferred onto Matrigel/mTesR1 conditions. iPSCs features were confirmed by microscopic observation and only colonies displaying typical iPSC-like colony morphology were selected and further confirmed by immunocytochemical analyses showing the expression of typical markers (Oct3/4, SOX2, NANOG, SSEA4, TRA1-60 and TRA1-81).
Pluripotency was also confirmed by the capacity of the chosen iPSC lines to give rise to all three germ layers under differentiating conditions. For this study, 2 validated iPSC clones were randomly chosen (one from each donor) and transferred into feeder-free conditions. In brief, human iPSCs were cultured in chemically defined growth media, StemMACS™ iPS-Brew XF medium (Miltenyi Biotec), on growth-factor-reduced Matrigel (BD Biosciences)-coated plates.
For iPSC maintenance/amplification, 70-80% confluent human iPSCs were treated with with an enzyme-free solution (hereafter referred as Gentle Dissociation Solution) and containing 0.5 mM EDTA (Life Technologies), D-PBS (Life Technologies) and 1.8 mg/mL NaCl (Sigma). iPSCs were incubated for 2 min in Gentle Dissociation Solution at 37°C and the colonies were dispersed to small clusters and lifted carefully using a 5 mL glass pipette at a ratio of 1:4.
When necessary, differentiated areas were removed from iPSC cultures prior to passaging in order to maintain the cultures in undifferentiated state prior to proceeding to their differentiation. Both iPSC lines were maintained in an incubator (37°C, 5% CO2) with medium changes every day.

Derivation of NPCs from iPSCs
Neural progenitor cell (NPC) were differentiated from hiPSCs by applying two different differentiation protocols. 1) Induction of NPCs was based on previous reports with slight modifications (2,3). One day before induction, hiPSCs were passaged onto growth-factor Advanced DMEM/F12, 50% Neurobasal, 1x N2, 1x B27, 2 mM GlutaMAX, 10 ng/mL hLIF, 3 μM CHIR99021, and 2 μM SB431542. Generated NPCs with this protocol were maintained on growth-factor-reduced matrigel in NSMM. NPCs were passaged once reaching 80% confluency using Accumax and seeded at about 20% confluency. Medium was changed every day. SMADi Neural Induction Kit + 10 μM μM Y-27632 and plated onto growth-factor-reducedcoated plates (320,000 cells cm−2). Cells were maintained in an incubator (37°C, 5% CO2) with medium changes every day and observed under a microscope on a daily basis. Between 6 and 9 days after induction, cells were harvested as single cells using Accumax, transferred into DMEM-F12 media, counted and centrifuged at 200×G for 5 min prior to be resuspended in STEMdiffTM SMADi Neural Induction Kit + 10 μM μM Y-27632 and plated onto growthfactor-reduced-coated plates (270,000 cells cm−2). Cells were maintained in an incubator (37°C, 5% CO2) with medium changes every day and observed under a microscope on a daily basis. 5 days post-seeding, cells were once more passaged following the same procedure. Finally, 5 days after the last passage (i.e. 16 to 19 days post-induction), differentiated cells were harvested as single cells using Accumax, transferred into DMEM-F12 media, counted and centrifuged at 200×G for 5 min prior to be resuspended in STEMdiffTM Neural Progenitor Medium (Stem Cell Technologies) and seeded onto growth-factor-reduced matrigel-coated plates (125,000 cells cm−2). iPSC-NPCs were maintained in an incubator (37°C, 5% CO2) with medium changes every day and passaged with Accumax when reaching 80-90% confluency. Of note, iPSC-NPCs between Passage 3 and 5 were used for further maturation into neurons.

Differentiation of hiPSC-NPCs into neurons
hiPSC-NPCs were differentiated into mature neurons with the BrainPhys TM Neuronal Medium Kit (Stem Cell Technologies) following manufacturer's instructions; which allows for the generation of mature neuronal cultures as previously described (4). In short, hiPSC-NPCs were harvested as single cells using Accumax, transferred into DMEM-F12 media, counted and centrifuged at 200×G for 5 min prior to be resuspended in supplemented BrainPhysTM Neuronal Medium (following manufacturer's recommendations) and plated onto Poly-L-Ornithin (10 µg/mL) /Laminin (4 µg/mL) -coated plates (50,000 cells cm −2 ). Cells ongoing neuronal differentiation/maturation were maintained in an incubator (37°C, 5% CO2) with half medium changes once a week. Cells were harvested at indicated time points throughout the study.

SH-SY5Y differentiation
SH-SY5Y were transferred onto 6-well or 24-well plate at a density of 5 x 10 4 and 1 x 10 4 cells/well respectively. Cell differentiation was performed in Neurobasal medium with 1% glutamine, 1% P/S, 1x B27 supplement (all Life Technologies), 50 ng/ml hBDNF (Miltenyi Biotec), 10 µM retinoic acid, 2mM dibutyryl AMPc, 20 mM KCl (all Sigma-Aldrich) as previously described (5). In contrast to currently used SH-SY5Y models, this improved model captures early neurodevelopmental processes with high fidelity. Differentiation medium was changed every day and cells were harvested at day 0, 1, 3, 5, 7 and 9 of differentiation in vitro (DIV) for RNA and/or protein analysis. For knockdown experiments, undifferentiated cells were incubated during 24 hours with siRNA before the initiation of differentiation and the differentiation was stop at day 5.
The final product cAB03 (i.e., pX459-pEF1alpha) was then obtained by SLIC method mixing the pX459 XbaI-EagI fragment and the EF1alpha PCR product as previously described (7). Therefore, cAB03 was used to produce two distinct plasmids, each containing a COSMOC specific single guide RNA (i.e., sgRNA containing crRNA + TracrRNA) sequence aiming at cutting a 140bp fragment portion within the exon1. To that end, CRISPR sgRNAs were designed using Crispor design tool (http://crispor.tefor.net) and cloned into cAB03 as previously described (6). For each of the two sgRNAs, a 20nt long COSMOC specific sequence was selected as follows: sgRNA1: GCTGGCCTGAAAGTGAAGAC; and sgRNA2: TTTAGCTATGTCTCGCGGAG. In short, each of the two selected COSMOC specific sequences was cloned by BbsI-mediated digestion of cAB03 followed by ligation using cohesive-end cloning. Plasmids were transformed into DH5alpha chemically-competent cells and individual clones were first screened by PCR to validate the insertion and ultimately validated by sequencing analysis.  Table 2, ddH20) and PCR reaction was performed using the following PCR conditions: 1min at 94°C for one cycle, then 10sec at 98°C and 1min at 55°C for 35 cycles and to finish, 5min at 68°C for one cycle.
Then, the PCR product was run on a 2% agarose gel electrophoresis to analyze the size of the amplicon for each individual clone (expected profiles: 809bp (Wild Type); ~699bp (Knock-Out presenting a 140bp deletion); two bands at 809bp and ~699bp (heterozygous lines)). Only those clones that were effectively Knock-Out for COSMOC were amplified.

Transfection of cells and Primers
Twenty-four hours after seeding, cells were transfected with Stealth RNAi™ siRNAs (Life Technologies) using Lipofectamine RNAimax (Life Technologies). Experiments using Stealth

RNA preparation, PCR and qPCR
Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manifacturer's instruction. Cytoplasmic/nuclear RNAs were fractioned and extracted using PARIS™ KIT (ThermoFisher Scientific). RNAs were reverse transcribed by M-MLV reverse transcriptase (Invitrogen) with random hexamers and PCR reactions were run with Taq DNA Polymerase (Invitrogen). Primers sequences are reported in Supplementary Table S2. Quantitative PCR experiments were carried out with the 7500 Fast Real-Time PCR system (ThermoFisher Scientific), using TaqMan™ Fast Universal PCR Master Mix and TaqMan™ Gene Expression Assays (ThermoFischer Scientific). Assays ID are reported in Supplementary   Table S3.

Oxidative stress induction
OSCs were cultured at a density of 2 x 10 6 cell/well onto 6-well plate. Twenty-four hours after seeding, cells were stressed for 2 or 4 hours in the presence of H2O2 (500µM) before RNA extraction.

ROS assay and flow cytometry analysis
Forty-eight hours after siRNAs transfection, intracellular ROS was measured by staining with CellRox® Deep Red Flow Cytometry Assay kit (Life Technologies). Stem cells were harvested and stressed with tert-butyl hydroperoxide (TBHP; 200 µM) at 37°C for 30 minutes. Cells were then treated with CellROX reagent (500 nM) at 37°C for 45 minutes. Staining was measured using FacsCanto cytometer (Becton Dickinson) and data were analyzed using FACS DIVA software.

Microarray gene expression
Stem cells from two different healthy individuals were collected 48 hours after treatment with siRNAs. RNA was extracted as described above. Genome-wide transcriptional profiling was performed by Human Exon 1.0 ST arrays (Affymetrix) following manufacturer's instructions.